Aminoquinoline derivatives and uses thereof

ABSTRACT

Described herein are aminoquinoline and aminoacridine based hybrids, pharmaceutical compositions and medicaments that include such aminoquinoline and aminoacridine based hybrids, and methods of using such compounds for diagnosing and/or treating infections, neurodegenerative diseases or disorders, inflammation, inflammation associated diseases and disorders, and/or diseases or disorders that are treatable with dopamine agonists such as the restless leg syndrome.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/382,727, filed on Sep. 3, 2014, which is a 35 U.S.C. § 371National Stage Entry application of International Application No.PCT/US2013/028329, filed Feb. 28, 2013, which designates the U.S., andwhich claims benefit under one or more of 35 U.S.C. § 119(a)-119(d) ofIndian Patent Application No. 661/DEL/2012, filed Mar. 7, 2012, thecontents of each of which are incorporated herein by reference in theirentirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant no. MH048866awarded by the National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application has been submittedelectronically via EFS-Web as an ASCII formatted sequence listing withthe file name: “Div_of_14/382,727_051058_076543_DIV.txt”, creation dateof Jan. 11, 2017 and a size of 8,494 bytes”. The sequence listingsubmitted via EFS-Web is part of the specification and is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates generally to aminoquinoline andaminoacridine based hybrids, pharmaceutical compositions and medicamentsthat include such aminoquinoline and aminoacridine based hybrids, andmethods of using such compounds for diagnosing and/or treatinginfections, neurodegenerative diseases or disorders, inflammation,and/or inflammation associated diseases and disorders.

BACKGROUND

PD, primarily caused by selective degeneration of midbrain dopamine(mDA) neurons, is the most prevalent movement disorder, affecting 1-2%of the global population over the age of 65¹⁻³ Currently availablepharmacological treatments (e.g., L-DOPA) are largely symptomatic andlose their efficacy over time, with accompanying severe side effectssuch as dyskinesia. Thus, there is an unmet clinical need to developmechanism-based and/or disease-modifying treatments^(2,3). The orphannuclear receptor Nurr1 (also known as NR4A2) is essential not only fordevelopment and maintenance of mDA neurons⁴⁻⁷ but also for theirprotection from inflammation-induced death⁸. Furthermore, previousstudies showed decreased Nurr1 expression in postmortem brains of PDpatients⁹ and functional mutant forms in rare familial PD cases¹⁰,strongly suggesting that Nurr1 is a promising target for the developmentof novel disease-modifying therapeutics for PD¹¹. Thus, there is a needin the art for agonists of Nurr1.

SUMMARY

In continuation of inventors efforts to develop new structurally diversemolecular scaffolds for the treatment of malaria [Rawat, D. S. Bioorg.Med. Chem. Lett. 2008, 18, 1446; Rawat, D. S. Bioorg. Med. Chem., 2009,17, 5632; Rawat, D. S. Eur. J. Med. Chem. 2011, 46, 2816; Manohar, S.,Khan, S. I., Rawat, D. S. Bioorg. Med. Chem. Lett. 2010, 20, 322;Manohar, S., Khan, S. I., Rawat, D. S. Chem. Biol. Drug Des. 2011, 78,124; Manohar, S., Rajesh, U. C., Khan, S. I., Tekwani, B. L., Rawat, D.S. ACS Med. Chem. Lett. 2012, 3, 555; and Manohar, S., Khan, S. I.,Rawat, D. S. Chem. Biol. Drug Des., Accepted, 2013 (DOI:10.1111/cbdd.12108)], an effort has been initiated to link4-aminoquinoline and pyrimidine entities together via flexible linker sothat molecule has enough flexibility to fit in the binding site of thetarget and as a result this kind of hybrid molecules may show betterantimalarial activity.

In one aspect, the disclosure provides a compound of formula (I), (II),(III), (IV), (V) (VI), (VII), (VIII), or (IX):

-   -   wherein:    -   X, Y, and Z are independently CR¹⁸, N, O, or S;    -   A and B are independently CR¹⁸ or N;    -   one of Z¹ and Z² is N and the other is CR¹⁴;    -   R and R¹⁰-R²⁸ independently H, linear or branched alkyl, linear        or branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted;    -   ----- forms a cyclyl, heterocyclyl, aryl, or heteroaryl, each of        which can be optionally substituted; and    -   L is a linker.

The compounds disclosed herein include pharmaceutically acceptablesalts, hydrates, solvates, esters, stereoisomer mixtures, andenantiomers thereof.

The disclosure also provides a pharmaceutical composition comprising acompound disclosed herein and a pharmaceutically acceptable diluent,carrier, or excipient.

In yet another aspect, the disclosure provides a method of treating adisease state when a decrease Nurr1 activity contributes to thepathology or symptomology of the disease, the method comprisingadministering a therapeutically effective amount of a disclosed hereinto a subject in need thereof.

In one aspect, the disclosure provides a method of treating aneurodegenerative disease or disorder in a subject, the methodcomprising administering a therapeutically effective amount of adisclosed herein to a subject in need thereof.

In yet another aspect, the disclosure provides a method of treating aneurodegenerative disease or disorder in a subject, the methodcomprising administering a therapeutically effective amount of adisclosed herein to a subject in need thereof.

In still another aspect, the disclosure provides a method of treatinginflammation or inflammation associated disorder in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of a compound described herein.

In yet still another aspect, the disclosure provides a kit that includesa compound disclosed herein together with instructions for administeringthe compound to subject in need thereof.

The disclosure further provides a method for causing differentiation ofa cell, e.g., a stem cell, into a dopaminergic neuron by contacting thecell with a compound disclosed herein.

The disclosure additionally features a method for treating aneurodegenerative disease or disorder in a subject, comprising:co-administering: (i) a composition containing stem cells into thesubject and (ii) a compound disclosed herein in an amount sufficient toinduce differentiation of the stem cells.

In some embodiments, the neurodegenerative disease is Parkinson'sdisease (PD).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the structures of amordiaquine (AQ) and chloroquine (CQ).

FIG. 2 shows structure of some exemplary aminoquinoline-pyrimidineanalogs according to embodiments of the compounds disclosed herein.

FIGS. 3-12 show exemplary embodiments of the compounds of the invention.

FIGS. 13A-13I show identification of amodiaquine (AQ) and chloroquine(CQ) as Nurr1 activators. (FIG. 13A) Chemical structures of three hitcompounds that activate Nurr1's transcriptional function. Notably, allcompounds contain an identical scaffold, 4-amino-7-chloroquinoline(highlighted by red color), strongly suggesting a structure-activityrelationship regarding Nurr1 activation. (FIG. 13B) AQ and CQ increasethe transcriptional activity of Nurr1-based reporter constructs:full-length Nurr1-dependent (fNurr; left panel) and Nurr1 LBD-dependent(right panel) transcriptional activities. The basal level oftranscriptional activity was normalized to 1. The bars representmeans±SEM from three independent experiments. (FIG. 13C) Effect of Nurr1specific siRNA on Nurr1 LBD's transactivation activity. A knock-down ofNurr1 expression by treatment with Nurr1-specific siRNA reduced thereporter gene activity of the Nurr1 LBD construct, suggesting that thisactivity is mediated through Nurr1. (FIG. 13D) Human neuroblastomaSK-N-BE(2)C cells were transfected with the N-terminal flag-tagged SRC1or SRC3 and the C-terminal myc-tagged full-length Nurr1. Twenty hoursafter transfection, cells were treated with 30 μM AQ or 100 μM CQ for 16hours. Cell lysates were immunoprecipitated (IP) using anti-flagantibodies and analyzed by immunoblotting (IB) using anti-mycantibodies. (FIG. 13E) Effect of SRC proteins on AQ and CQ-induced Nurr1transactivation. SK-N-BE(2)C cells were transfected with VP16-SRC1 orVP16-SRC3, an effector plasmid (pSV GAL_((DBD))-N_((LBD))), and areporter plasmid (p8xUAS-Luc), and were treated with 30 μM AQ or 100 μMCQ for 16 hours. The fold induction was derived by comparing eachluciferase activity to basal level obtained by non-SRC transfection.(FIG. 13F) AQ showed highly specific interaction with Nurr1 LBD asevaluated by surface plasmon resonance (SPR) analysis. The sensorgramswere obtained from injection of a series of concentrations of AQ overimmobilized Nurr1-LBD and RXR-LBD. The concentrations (04) are shownnext to the arrows. (FIG. 13G) Fluorescence spectra of Nurr1-LBD andRXR-LBD in the presence of increasing concentrations of AQ followingexcitation at 280 nm in PBS (pH 7.2). The arrows indicate signal changesas AQ concentration increases (0, 0.1 nM, 1 nM, 10 nM, 100 nM and 104).(FIG. 13H) Saturation binding of the recombinant Nurr1-LBD protein using[³H]-CQ. Nurr1-LBD protein was incubated for overnight at 4° C. withincreasing concentrations (3.9, 7.8, 15.5, 31, 62.5, 125, 250, 500, 1000nM) of [³H]-CQ. Non-specific binding was determined in the presence of a1000-fold molar excess of unlabeled CQ. Specific binding was calculatedas the difference of total and non-specific binding. The insertindicates Scatchard analysis of the specific binding. (FIG. 13I)Competition of AQ, CQ and primaquine (PQ) with [³H]-CQ for binding tothe Nurr1-LBD. Increasing concentrations of unlabeled AQ, CQ or PQ wereincubated with 500 nM [³H]-CQ and Nurr1-LBD. The data represent theaverage of triplicate from three independent experiments. All graphs,K_(d), K_(i), and B_(max) values were generated using the non-linearregression program Prism version 5.02.

FIGS. 14A-14G show functional effects of AQ and CQ. (FIGS. 14A and 14B)AQ stimulated the generation and gene expression of DA neurons fromneural progenitors isolated from E14.5 rat cortex in a dose-dependentmanner. Cortical precursors were transduced with Nurr1-expressingretrovirus and the TH+ cells were visualized by immunostaining againstTH protein. Differentiation was induced by withdrawal of bFGF a dayafter viral infection and AQ was added for 2 hours during thedifferentiation period. Immunocytochemical analyses for TH (FIG. 14A)and yields of TH+/DAPI cells (FIG. 14B) for each treatment group wereobtained following in vitro differentiation for 3 d and 9 d.Significantly different from control in 3 independent cultures. (FIG.14C) Schematic representation of the location of primers used in the ratTH promoter (upper panel). Primer sequences are shown in materials andmethods. ChIP assay shows AQ- or CQ-dependent Nurr1 recruitment to theTH promoters NL1 and NL3. Rat PC12 cells were treated with 20 μM AQ or70 μM CQ for 15 hrs. ChIP assay was carried out with IgG or α-Nurr1(E-20) antibody. *p<0.05 and **p<0.005 versus untreated. Results areexpressed as the average of three independent experiments. Error barsrepresent standard deviations. (FIGS. 14D-14F) Primary cultures of ratmesencephalic DA neurons were treated with 20 μM 6-OHDA for 24 hr in thepresence or absence of 50 μM AQ and 20 μM CQ. (FIG. 14D) The number ofTH-positive neurons and (FIG. 14E) the rate of [³H]DA uptake weremeasured. (FIG. 14F) Cell survival was measured by the MTT reductionassay in DA cells treated with 6-OHDA alone or in combination with AQ.Values from each treatment were expressed as a percentage of untreatedcontrol for the MTT assay. (FIG. 14G) AQ suppresses LPS-inducedexpression of proinflammatory cytokines. Primary microglia from P1 ratbrains were treated with 1 ng/ml LPS for 4 hrs in the presence orabsence of AQ (10 and 20 μM). Levels of mRNA expression were analyzed byquantitative real-time PCR and normalized with GAPDH. Each barrepresents means±SEM of n=4-5. *p<0.05, **p<0.01, ***p<0.001, comparedto the LPS only-treated group.

FIG. 15A-15F show effects of AQ treatments on 6-OHDA-lesioned rat modelof PD. (FIG. 15A) Schematic representation of the administration of AQto 6-OHDA lesioned rats. Unilateral striatal 6-OHDA lesioned rats weretreated with AQ or saline for 2 weeks, starting from 1 day prior tolesioning. The grey shade indicates L-DOPA treatment for 2 weeks ascontrol of AIMs test. (FIG. 15B) Amphetamine-induced rotational test wasperformed at 4 and 6 weeks (right panel) after 6-OHDA lesion. There wassignificant difference in the number of amphetamine-induced rotationalbehavior between the two groups at 4 and 6 weeks. (FIG. 15C) TH⁺ fibersand neurons were plentiful in the ST and SN of normal site whereas theirmarked depletion was observed in the right ST and SN by 6-OHDAlesioning. In contrast, abundant TH-immunopositive cells were spared notonly in the striatum and but also in the SN in AQ-treated group, whenexamined at 6 weeks post lesion. Scale bar: black=200, while=100, red=20μm. (FIG. 15D) Stereological counting in AQ-treated rats show thatapproximately 60% of TH⁺ neurons are spared in the lesion side, whereasit remained about 10% in saline-treated animals. (FIG. 15E) Iba1+microglial activation by 6-OHDA lesion was dramatically decreased by AQtreatment. Scale bar: while=50, red=10 μm. (FIG. 15F) Treatment of6-OHDA-lesioned rats with L-DOPA, but not with AQ, exhibited severe sideeffects, as measured by AIMs scores. Rats were monitored for four typesof AIMs (Axial, Forelimb, Orolingual, and Locomoter AIMS) at 2 and 6weeks post lesioning. Each AIM behavior was monitored and scored with0-4 scale and summed. Bars represent the mean±SEM (^(#)p<0.06, *p<0.01,**p<0.0003, ***p<0.0001).

FIGS. 16A-16D show that AQ and CQ activate the transcriptional activityof full-length Nurr1 (FIGS. 16A and 16B) and Nurr1 LBD (FIGS. 16C and16D in a dose-dependent manner.

FIG. 17 shows the effect of Nurr1-specific siRNA on the transcriptionalactivity of full-length Nurr1.

FIG. 18 show that structure activity relationship (SAR) analysisindicates that 4-amino-7-chloroquinoline can be an essential feature fortransactivation function of Nurr1. The inventors examined the functionof related quinoline compounds for potential activation of Nurr1. Noneof the quinoline compounds (without 4-amino-7-chloroquinoline) testedhere exhibited any detectable transactivation of the Nurr1 LBD over awide range of concentrations. Bars represent means±SEM from threeindependent experiments.

FIG. 19 shows the target selectivity of AQ for LBDs of various NRs. 30μM AQ robustly activates LBD function of Nurr1, but not other NRs testedhere, indicating a high specificity. The positive reactivity of these NRconstructs was confirmed by the known activators (2 nM dexamethasone, 20nM retinoic acid, 2 μM GW3965, 50 nM GW7647, and 5 nM GW1929 for GR,RXR, LXR, PPARα, and PPARγ, respectively). The basal level oftranscriptional activity was normalized to 1. Bars represent means±SEMfrom three independent experiments. (Nur77; NR4A1; nuclear receptorsubfamily 4 group A member 1, GR; glucocorticoid receptor, LXR; liver Xreceptor-alpha, RXR; retinoid X receptor-alpha, PPARα; peroxisomeproliferator-activated receptor-alpha, PPARγ; peroxisomeproliferator-activated receptor-gamma)

FIGS. 20A-20C shows that AQ does not interact with Nur77-LBD. (FIG. 20A)AQ showed no interaction with Nur77-LBD as evaluated by SPR analysis.Sensorgrams were obtained from injection of a series of AQconcentrations over immobilized Nur77-LBD proteins. (FIGS. 20B and 20C)Fluorescence spectra of Nur77-LBD in the presence of increasingconcentrations of AQ (0, 0.1 nM, 1 nM, 10 nM, 100 nM and 1 μM) (FIG.20B), or of RXR-LBD in the presence of increasing concentration of 9-cisretinoic acid (0, 0.1 nM, 1 nM, 10 nM, 100 nM and 1 μM) as a positivecontrol (FIG. 20C) following the excitation at 280 nm at PBS (pH 7.2).

FIG. 21 shows results of real time PCR analysis showing that AQtreatment enhances expression of the mDA-specific genes (e.g., tyrosinehydroxylase (TH), dopamine transporter (DAT), vesicular monoaminetransporter (VMAT), and aromatic amino acid decarboxylase (AADC)) duringin vitro differentiation of neural stem cells. mRNAs from each treatmentgroup were obtained following in vitro differentiation for 9 d.

FIGS. 22A and 22B shows effect of AQ and CQ on expression ofpro-inflammatory cytokine genes. (FIG. 22A) AQ suppresses LPS-inducedexpression of pro-inflammatory cytokines in murine microglial BV-2 cellthat treated with 10 ng/ml LPS for 4 hrs in the presence or absence ofAQ (10 and 15 μM). (FIG. 22B) CQ suppresses LPS-induced expression ofproinflammatory cytokines. Primary microglia from P1 rat brains weretreated with 10 ng/ml LPS for 4 hrs in the presence or absence of 10 μMCQ. Levels of mRNA expression were analyzed by quantitative real-timePCR and normalized with GAPDH. Each bar represents means±SEM of n=6.*p<0.05, **p<0.01,***p<0.001, compared to the LPS only-treated group.

FIG. 23 shows that spared TH⁺ cells in the lesioned hemisphere of 6-OHDArats following AQ treatments co-expressed mature DA neuronal markerssuch as FoxA2 and AADC. Scale bar=100 μm.

FIGS. 24A-24J. AQ reduces microglial activation in 6-OHDA-injected ratbrains. Rats were administered saline (FIGS. 24A, 24B, 24E, and 24F) orAQ (FIGS. 24C, 24D, 24G, and 24H) for 2 weeks, then were sacrificed andbrains sectioned. Brain sections including the SN (FIGS. 24A-24D) andSTR regions (FIGS. 24E-H) were immunostained with anti-Iba-1 antibodyand Iba-1⁺ cells were counted in both lesion and intact sides. FIGS.24I-24J are bargraphs showing percentage of Iba-1+ cells in lesionedside/Iba-1+ cells in intact side for SN and STR regions respectively.Data represent the mean±SEM. (**p<0.01, ***p<0.001). Scale bar=200 μm.

FIGS. 25A-25D show that AQ treatment to ak/ak mice resulted infunctional improvements in nigrostriatal-specific motor behavior testssuch as Pole Test (FIG. 25A), Beam Test (FIG. 25B), Cylinder Test (FIG.25C), and Rotarod Test (FIG. 25D). L-Dopa treatment was used as positivecontrol. Bars represent the mean±SEM (*p<0.05, **p<0.01, ***p<0.001).

FIG. 26 shows that AQ reduces inflammation in the SN of ak/ak mice.ak/ak mice were administered saline or AQ for 2 weeks. Mice weresacrificed and their brains sectioned. Sections of SN regions from wildtype and ak/ak mice were immunostained with anti-CD11b and anti-THantibodies. Stereological counting of CD11b+ microglia was performed inthe SN area of ak/ak mice and presented as a percentage of the cellnumber in the wild type. Data represent the mean±SEM (**p<0.01).

FIG. 27 show that some derivatives of CQ retaining the 4-amino-7-chloroquinolone structure, as shown in FIG. 2, exhibit significantly higherefficacy than the starting compound, CQ. While the EC₅₀ of CQ isapproximately 60 μM (see FIG. 16), these four derivatives' EC₅₀ is lessthan one-tenth of CQ.

DETAILED DESCRIPTION

One object of the invention is to develop a method for the preparationaminoquinoline based hybrids in which 4-aminoquinoline,8-aminoquinoline, mefloquine, amidoquine, are covalently attached topyrimidine, pyrazine, triazine C7/C5 curcuminods, heteroaromatics,aromatics, amino acids, peptides, sugar, steroids, or any otherantimalarial pharmacophores and all related possible combinations.Another object of the invention is to synthesis aminoquinoline basedhybrids in which aminoquinoline is attached with various pharmacophoreswhich are known for their antimalarial activity. An additional object ofthis invention is to use these aminoquinoline based hybrids for thetreatment of malarial using any delivery agent. Yet another object ofthe invention is to use these aminoquinoline based hybrids for treatmentof disease when a decrease Nurr1 activity contributes to the pathologyor symptomology of the disease. Still another object of the invention isto use these aminoquinoline based hybrids for treating aneurodegenerative disease or disorder. Still another aspect of theinvention is using these aminoquinoline based hybrids for treatinginflammation or inflammation associated disorder. Yet another object ofthe invention is to attach these aminoquinoline based hybrids tobiocompatible polymers, dendrimers and nano materials for the treatmentof infectious diseases. Further object of the invention is to use thesecompounds for the treatment of antimalarial as such or in combination ofany other antimalarial drugs. The foregoing has outlined some of thepertinent objectives of the invention. These objectives should beconstrued to be merely illustrative of some of the more prominentfeatures and applications of the intended invention. Many otherbeneficial results can be obtained by applying the disclosed inventionin a different manner or modifying the invention within the scope ofdisclosure. Accordingly, other objectives and a full understanding ofthe invention and the detailed description of the preferred embodimentin addition to the scope of invention are to be defined by the claims.

As will be appreciated by one of skill in the art, the compounds offormula (I)-(V) disclosed herein are based on the 4-aminoquinoline orthe 8-aminoquinoline skeleton. Accordingly, in some embodiments, Z¹ is Nand Z² is CR¹⁴ (e.g., the 4-aminoquinoline skeleton). In some otherembodiments, Z¹ is CR¹⁴ and Z² is N (e.g., the 8-aminoquinolineskeleton).

Without limitations, variables R¹²-R¹⁷ can all be the same, alldifferent or any combinations of same and different. In one non-limitingexample, at least one (e.g., one, two, three, four, five, or all six) ofR¹²-R¹⁷ can be H. In some embodiments, R¹², R¹³, R¹⁵, and R¹⁷ are all H.In some embodiments, R¹², R¹³, R¹⁵, and R¹⁷ are all same; Z¹ is N and Z²is CH. In one embodiment, R¹², R¹³, R¹⁵, and R¹⁷ are all H; Z¹ is N andZ² is CH.

In another non-limiting example, at least one (e.g., one, two, three,four, five, or all six) of R¹²-R¹⁷ is not H. In some embodiments, atleast one (e.g., one, two, three, four, five, or all six) of R¹²-R¹⁷ ishalogen, alkyl, cyclyl, hydroxyl, alkoxy, thio, alkylthio, SO₂R¹⁰,amino, alkylamino, amino acid, or carbohydrate.

In some embodiments, at least one (e.g., one, two, three, four, five, orall six) of R¹²-R¹⁷ is halogen, e.g., Br, Cl, or F. In some embodiments,R¹⁶ is halogen, e.g., Br, Cl, or F. In some other embodiments, R¹⁷ ishalogen, e.g., Br, Cl, or F.

In some embodiments, at least one (e.g., one, two, three, four, five, orall six) of R¹²-R¹⁷ is C₁-C₆ alkyl, which can be optionally substituted.In some embodiments, R¹⁶ is C₁-C₆ alkyl, which can be optionallysubstituted. In some other embodiments, R¹⁷ is C₁-C₆ alkyl, which can beoptionally substituted.

In some embodiments, at least one (e.g., one, two, three, four, five, orall six) of R¹²-R¹⁷ is CO₂H. In some embodiments, R¹⁶ is CO₂H. In someother embodiments, R¹⁷ is CO₂H.

In some embodiments, at least one (e.g., one, two, three, four, five, orall six) of R¹²-R¹⁷ is hydroxyl. In some embodiments, R¹⁶ is hydroxyl.In some other embodiments, R¹⁷ is hydroxyl.

In some embodiments, at least one (e.g., one, two, three, four, five, orall six) of R¹²-R¹⁷ is alkoxy. In some embodiments, R¹⁶ is alkoxy. Insome other embodiments, R¹⁷ is alkoxy.

In some embodiments, at least one (e.g., one, two, three, four, five, orall six) of R¹²-R¹⁷ is thiol or alkylthio. In some embodiments, R¹⁶ isthiol or alkylthio. In some other embodiments, R¹⁷ is thiol oralkylthio.

In some embodiments, at least one (e.g., one, two, three, four, five, orall six) of R¹²-R¹⁷ is amino or alkylamino. In some embodiments, R¹⁶ isamino or alkylamino. In some other embodiments, R¹⁷ is amino oralkylamino.

In some embodiments, at least one (e.g., one, two, three, four, five, orall six) of R¹²-R¹⁷ is cyano. In some embodiments, R¹⁶ is cyano. In someother embodiments, R¹⁷ is cyano.

In some embodiments, R¹⁸ is a hydrogen, halogen, cyano, nitro, C₁-C₆alkyl, or optionally substituted phenyl.

In some embodiments, R¹⁸ is hydrogen. In some embodiments, R¹⁸ is a4-substituted phenyl. In one embodiment R¹⁸ is

wherein R¹⁰⁴ is linear or branched alkyl, linear or branched alkenyl,linear or branched alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl,halogen, alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,alkylamino, thiol, or alkylthio, each of which can be optionallysubstituted. In some embodiments, R¹⁰⁴ is Br.

In some embodiments, R¹², R¹³, R¹⁵, and R¹⁷ are H; R¹⁶ is chloro; Z¹ isN; and Z² is CH. In some other embodiments, R¹², R¹³, R¹⁵, R¹⁶, and R¹⁷are H, Z¹ is CH; and Z² is N.

In still some other embodiments, R¹², R¹³, R¹⁵, R¹⁶, and R¹⁷ are H, Z¹is N; and Z² is CH.

Without limitations, variables R²¹-R²⁸ can all be the same, alldifferent or any combinations of same and different. In one non-limitingexample, at least one (e.g., one, two, three, four, five, six, seven, orall eight) of R²¹-R²⁸ can be H. In some embodiments, R²¹, R²², R²⁴, R²⁵,R²⁷ and R²⁸ are all H. In another non-limiting example, at least one(e.g., one, two, three, four, five, six, seven, or all eight)) ofR²¹-R²⁸ is not H. In some embodiments, at least one (e.g., one, two,three, four, five, six, seven, or all eight) of R²¹-R²⁸ is halogen,alkyl, cyclyl, hydroxyl, alkoxy, thio, alkylthio, SO₂R¹⁰, amino,alkylamino, amino acid, or carbohydrate.

In some embodiments, at least one (e.g., one, two, three, four, five,six, seven, or all eight) of R²¹-R²⁸ is halogen, e.g., Br, Cl, or F. Insome embodiments, R²³ is halogen, e.g., Br, Cl, or F.

In some embodiments, at least one (e.g., one, two, three, four, five,six, seven, or all eight) of R²¹-R²⁸ is C₁-C₆ alkoxy. In someembodiments, at least one (e.g., one, two, three, four, five, six,seven, or all eight) of R²¹-R²⁸ is methoxy. In some embodiments, R²⁶ isC₁-C₆alkoxy. In one embodiment, R²⁶ is methoxy.

In one embodiment, R²¹, R²², R²⁴, R²⁵, R²⁷ and R²⁸ are all H; R²³ is Cl;and R²⁶ methoxy.

In the ring comprising the variables X, Y, and Z, the variables X, Y,and Z can all be same, all different, or any combination of same anddifferent, e.g., all three can be same, two same and one different, orall three are different. For example, X, Y, and Z call can be the same;X and Y can be same while Z is different; X and Z can be the same whileY is different; Y and Z can be the same while X is different; or X, Y, Zare all different.

In some embodiments, at least one (e.g., one, two, or all three) of X,Y, and Z are CH. In some embodiments, X, Y, and Z are all CH. In someembodiments, at least one (e.g., one, two, or all three) of X, Y, and Zare N. Various combinations of X, Y, and Z variables include: X, Y, andZ all are N; X and Y are N, and Z is CH; X and Z are N, and Y is CH; Xis N, and Y and Z are CH; X is CH, and Y and Z are N; X and Z are CH,and Y is N; X and Y are CH, and Z is N; and X, Y, and Z all are CH.Thus, the compounds disclosed herein can comprise any one of the variouscombinations disclosed in this paragraph.

In the ring comprising variables A and B, the variables A and B can bethe same or different. In some embodiments, A is N and B is CH; A is CHand B is N; or A and B both are N.

In some embodiments, R is H, C₁-C₆alkyl, halogen, heterocyclyl, amino,arylamino, cyclylamino, and alkylamino, each of which can be optionallysubstituted. In one embodiment, R is methyl.

Without limitations, variables R¹⁰ and R¹¹ can be same or different. Insome embodiments, R¹⁰ and R¹¹ can be selected independently from thegroup consisting of H, C₁-C₆alkyl, —NH(CH₂)x-OH (wherein x is an integerfrom 0 to 10), halogen, heterocyclyl, amino, arylamino, cyclylamino, andalkylamino, each of which can be optionally substituted.

In some embodiments, R¹⁰ can be selected from the group consisting of2-(7-chloro-4-quinolinylamino)-ethyl amino; 2-alkylaminoethyl amino;2-aminoethyl amino; 2-hydroxyethyl amino; 2-methylphenyl amino;3,5-dimethoxyphenyl amino; 3-alkylaminopropyl amino; 3-aminopropylamino; 3-hydroxypropyl amino; 4-alkylaminobutyl amino; 4-aminopropylamino; 4-ethylphenyl amino; 4-ethylpiperazinyl; 4-bromophenyl amino;4-chlorophenyl amino; 4-fluorophenyl amino; 4-hydroxybutyl amino;4-methoxyphenyl amino; 4-methylphenyl amino; 4-methylpiperazinyl;4-morpholinyl; 5-hydroxypentyl amino; 6-hexylapentyl amino;alkylaminobutyl amino; aminomethyl amino; Chloro; cycloheptylamino;cyclohexylamino; cyclopenylamino; diethylamino; dimethylamino; hydrogen;hydroxymethyl amino; methyl; phenylamino; piperazinyl; and piperidinyl,each of which can be optionally substituted.

In some embodiments, R¹¹ can be selected from the group consisting of2-(7-chloro-4-quinolinylamino)-ethyl amino; 2-alkylaminoethyl amino;2-aminoethyl amino; 2-hydroxyethyl amino; 2-methylphenyl amino;3,5-dimethoxyphenyl amino; 3-alkylaminopropyl amino; 3-aminopropylamino; 3-hydroxypropyl amino; 4-alkylaminobutyl amino; 4-aminopropylamino; 4-ethylphenyl amino; 4-ethylpiperazinyl; 4-bromophenyl amino;4-chlorophenyl amino; 4-fluorophenyl amino; 4-hydroxybutyl amino;4-methoxyphenyl amino; 4-methylphenyl amino; 4-methylpiperazinyl;4-morpholinyl; 5-hydroxypentyl amino; 6-hexylapentyl amino;alkylaminobutyl amino; aminomethyl amino; Chloro; cycloheptylamino;cyclohexylamino; cyclopenylamino; diethylamino; dimethylamino; hydrogen;hydroxymethyl amino; methyl; phenylamino; piperazinyl; and piperidinyl,each of which can be optionally substituted.

In some embodiments, R¹⁰ and R¹¹ can be selected independently from thegroup consisting of 2-(7-chloro-4-quinolinylamino)-ethyl amino;2-alkylaminoethyl amino; 2-aminoethyl amino; 2-hydroxyethyl amino;2-methylphenyl amino; 3,5-dimethoxyphenyl amino; 3-alkylaminopropylamino; 3-aminopropyl amino; 3-hydroxypropyl amino; 4-alkylaminobutylamino; 4-aminopropyl amino; 4-ethylphenyl amino; 4-ethylpiperazinyl;4-bromophenyl amino; 4-chlorophenyl amino; 4-fluorophenyl amino;4-hydroxybutyl amino; 4-methoxyphenyl amino; 4-methylphenyl amino;4-methylpiperazinyl; 4-morpholinyl; 5-hydroxypentyl amino;6-hexylapentyl amino; alkylaminobutyl amino; aminomethyl amino; Chloro;cycloheptylamino; cyclohexylamino; cyclopenylamino; diethylamino;dimethylamino; hydrogen; hydroxymethyl amino; methyl; phenylamino;piperazinyl; and piperidinyl, each of which can be optionallysubstituted.

In some embodiments, one of R¹⁰ and R¹¹ is 4-morpholinyl and the otheris 3-hydroxypropyl amino; one is 4-morpholinyl and the other is2-hydroxyethyl amino; one is 4-morpholinyl and the other is3,5-dimethoxyphenyl amino; one is 4-morpholinyl and the other is4-ethylphenyl amino; one is 4-morpholinyl and the other is4-fluorophenyl amino; one is 4-morpholinyl and the other is4-hydroxybutyl amino; one is 4-morpholinyl and the other is4-methoxyphenyl amino; one is 4-morpholinyl and the other isphenylamino; one is 4-piperidinyl and the other is phenylamino; one ischloro and the other is 2-(7-chloro-4-quinolinylamino)-ethyl amino; oneis chloro and the other is 4-ethylpiperazinyl; one is chloro and theother is 4-ethylpiperazinyl; one is chloro and the other is4-methylpiperazinyl; one is chloro and the other is 4-methylpiperazinyl;one is chloro and the other is 4-morpholinyl; one is chloro and theother is diethylamino; one is chloro and the other is piperidinyl; oneis chloro and the other one is phenyl amino; one is hydrogen and theother is 4-methoxyphenyl amino; one is hydrogen and the other is2-hydroxyethyl amino; one is hydrogen and the other is3,5-dimethoxyphenyl amino; one is hydrogen and the other is3-hydroxypropyl amino; one is hydrogen and the other is 4-bromophenylamino; one is hydrogen and the other is 4-chlorophenyl amino; one ishydrogen and the other is 4-fluorophenyl amino; one is hydrogen and theother is 4-hydroxybutyl amino; one is hydrogen and the other is4-methylphenyl amino; one is hydrogen and the other is 4-morpholinyl;one is hydrogen and the other is 5-hydroxypentyl amino; one is hydrogenand the other is 6-hydroxyhexyl amino; one is hydrogen and the other ischloro; one is hydrogen and the other is phenyl amino; one is hydrogenand the other is piperidinyl; one is methyl and the other is4-methoxyphenyl amino; one is methyl and the other is 2-hydroxyethylamino; one is methyl and the other is 3,5-dimethoxyphenyl amino; one ismethyl and the other is 3-hydroxypropyl amino; one is methyl and theother is 4-bromophenyl amino; one is methyl and the other is4-chlorophenyl amino; one is methyl and the other is 4-ethylpiperazinyl;one is methyl and the other is 4-fluorophenyl amino; one is methyl andthe other is 4-hydroxybutyl amino; one is methyl and the other is4-methylphenyl amino; one is methyl and the other is4-methylpiperazinyl; one is methyl and the other is 4-morpholinyl; oneis methyl and the other is 5-hydroxypentyl amino; one is methyl and theother is 6-hydroxyhexyl amino; one is methyl and the other is chloro;one is methyl and the other is phenyl amino; one is methyl and the otheris piperidinyl; one is piperidinyl 2-hydroxyethyl amino; one ispiperidinyl and the other is 3,5-dimethoxyphenyl amino; one ispiperidinyl and the other is 3-hydroxypropyl amino; one is piperidinyland the other is 4-ethylphenyl amino; one is piperidinyl and the otheris 4-fluorophenyl amino; one is piperidinyl and the other is4-hydroxybutyl amino; or one is piperidinyl and the other is4-methoxyphenyl amino.

In some embodiments, both R¹⁰ and R¹¹ are chloro; both are phenylamino;both are 4-morpholinyl; both are 2-methylphenyl amino; both arecyclohexyl amino; or both are hydrogen.

In some embodiments, one of R¹⁰ and R¹¹ is hydrogen or methyl and theother is cycloproylamine; cyclopentylamine; cyclohexylamine;cycloheptylamine; 1-adamantylamine; pyrrolidinyl; 4-thiomorpholinyl;4-alkyl-piperazinyl; pyrazolyl; 1H-pyrazolyl; imidazolyl; N-imidazolyl;3,5-dimethyl-pyrazolyl; or 3,5-dimethyl-1H-pyrazolyl.

In some embodiments, one of R¹⁰ and R¹¹ is

wherein q is an integer from 0 to 20. In some embodiments, q is 0, 1, 2,3, 4, 5, or 6. In one embodiment, q is 1, 2, 3, or 4.

In some embodiments, one of R¹⁰ and R¹¹ is H or methyl and the other is

wherein q is an integer from 0 to 20. In some embodiments, q is 0, 1, 2,3, 4, 5, or 6. In one embodiment, q is 1, 2, 3, or 4.

In some embodiments, one of R¹⁰ and R¹¹ is

In some embodiments, one of R¹⁰ and R¹¹ is H, methyl or chloro and theother is

In some embodiments, one of R¹⁰ and R¹¹ is

wherein R¹⁰⁰ and R¹⁰¹ are independently amino, alkylamino, cycloamino,heterocyclylamino, arylamino, heteroarylamino, or heterocyclyl linked bya ring nitrogen, each of which can be optionally substituted.

In some embodiments, one of R¹⁰ and R¹¹ is H, methyl or chloro and theother is

wherein R¹⁰⁰ and R¹⁰¹ are independently amino, alkylamino, cycloamino,heterocyclylamino, arylamino, heteroarylamino, heterocyclyl linked by aring nitrogen, or amino acid, each of which can be optionallysubstituted. In some embodiments, R¹⁰⁰ and R¹⁰¹ both are same. In someembodiments, R¹⁰⁰ and R¹⁰¹ are different.

In some embodiments, R¹⁸ is optionally substituted aryl. For example,R¹⁸ can be an optionally substituted phenyl. In some embodiments, R¹⁸ is4-bromophenyl.

In one embodiment, one of R¹⁰ and R¹¹ is

wherein R¹⁰³ is amino, alkylamino, cycloamino, heterocyclylamino,arylamino, heteroarylamino, heterocyclyl linked by a ring nitrogen, oramino acid, each of which can be optionally substituted. In oneembodiment, R¹⁰³ is

wherein Z¹⁰³ is CH₂, O, NH, N-methyl, N-ethyl, or S.

In some embodiments, R¹¹ is

In some embodiments, the compound of formula (I) is of formula (IA):

wherein the variables are as defined above.

In some embodiments, the compound of formula (I) is of formula (IB),(IC), (ID) or (IE):

wherein t is an integer from 0 to 20, and the other variables are asdefined above.

In some embodiments, t is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

In some embodiments, the compound of formula (I) is of formula (IF) or(IG):

wherein a and c are independently an integer from 0 to 20; c is aninteger from 0 to 5; R^(p) is absent or linear or branched alkyl, linearor branched alkenyl, linear or branched alkynyl, cyclyl, heterocyclyl,aryl, heteroaryl, halogen, alkoxy, nitro, cyano, carbonyl, carboxy,hydroxyl, phenoxy, amino, alkylamino, thiol, sulfinyl, sulfonyl,thiocarbonyl, or alkylthio, each of which can be optionally substituted;and the other variables are as defined above.

In some embodiments, a is 0, 1, 2, 3, 4, 5, or 6.

In some embodiment, b is 0, 1, or 2.

In some embodiments, c is 0, 1, 2, 3, 4, 5, or 6.

In some embodiments of formula (IF) or (IG), R¹⁰ is hydrogen or methyl.

In some embodiments of formula (IF) or (IG), R^(p) is absent. In someother embodiments, R^(p) is methyl, bromo, chloro, fluoro, or methoxy.

In some embodiments, a compound of formula (IV) is of formula (IVA)

wherein the variables are as defined above.

In some embodiments, a compound of formula (V) is of formula (VA):

wherein variable are as defined above.

In some embodiments, a compound of formula (VI) is of formula (VIA) or(VIB):

wherein d is an integer from 0 to 20; and the other variables are asdefined above.

In some embodiments, d is 0, 1, 2, 3, 4, 5, or 6.

As used herein, the term “linker” refers to an organic moiety thatconnects two parts of a compound. Linkers typically comprise a directbond or an atom such as oxygen or sulfur, a unit such as NR^(N′), C(O),C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R^(N′))₂, C(O), cleavable linkinggroup, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclic; where R^(N′) ishydrogen, acyl, aliphatic or substituted aliphatic.

The exact structure and size of the linker can vary considerably, andmany variations of the linker are within the scope of the inventionsdisclosed herein. As used herein, the “linker” can comprise a widevariety of structures. In many embodiments it is desirable that thelinker be of sufficient size and character that it provides some spaceand/or flexibility in the connection between the two parts of themolecule the linker is connecting together, but does not become of suchhigh molecular weight so as to impair the water solubility ortrans-membrane absorbability of the resulting compounds.

Accordingly, in some embodiments, the linker is selected from the groupconsisting of branched or linear alkylene, branched or linearalkenylene, branched or linear alkynylene, cyclyl, heterocyclyl, aryl,heteroaryl, O, NH, S, S, SS, SO₂, O-alkylene, NH-alkylene, S-alkylene,amino acid, carbohydrate, and any combinations thereof.

In some embodiments, the linker is —(CH₂)_(n)— or —CH(CH₃)—(CH₂)_(n)—,wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20. In some embodiments, n is 1. In some embodiments, nis 2. In some other embodiments, n is 3. In yet some other embodiment, nis 4. In yet still some other embodiments, n is 5. In some otherembodiments, n is 6. In still some other embodiments, n is 7. In oneembodiment, n is 8.

In some embodiments, the linker is

wherein m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20. In some embodiments, m is 0. In some otherembodiments, m is 1. In yet some other embodiment, m is 2. In yet stillsome other embodiments, m is 3.

In some embodiments, the linker is

wherein p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20. In some embodiments, p is 0. In some otherembodiments, p is 1. In yet some other embodiment, p is 2. In yet stillsome other embodiments, p is 3.

Additional exemplary linkers that can be used are shown in Table 1.

TABLE 1 Examples of hetero-bifunctional cross linking agents.Hetero-Bifunctional Cross Linking Agents Spacer Arm Length afterAdvantages and crosslinking Linker Reactive Toward Applications(angstroms) SMPT Primary amines Greater stability 11.2 Sulfhydryls SPDPPrimary amines Thiolation 6.8 Sulfhydryls Cleavable cross- linkingLC-SPDP Primary amines Extended spacer arm 15.6 Sulfhydryls Sulfo-LC-Primary amines Extended spacer arm 15.6 SPDP Sulfhydryls Water solubleSMCC Primary amines Stable maleimide 11.6 Sulfhydryls reactive groupEnzyme-antibody conjugation Hapten-carrier protein conjugationSulfo-SMCC Primary amines Stable maleimide 11.6 Sulfhydryls reactivegroup Enzyme-antibody conjugation MBS Primary amines Enzyme-antibody 9.9Sulfhydryls conjugation Hapten-carrier protein conjugation Sulfo-MBSPrimary amines Water soluble 9.9 Sulfhydryls SIAB Primary aminesEnzyme-antibody 10.6 Sulfhydryls conjugation Sulfo-SIAB Primary aminesWater soluble 10.6 Sulfhydryls SMPB Primary amines Extended spacer arm14.5 Sulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary aminesExtended spacer arm 14.5 Sulfhydryls Water-soluble EDC/Sulfo- Primaryamines Hapten-carrier 0 NHS Carboxyl groups conjugation ABHCarbohydrates Reacts with sugar 11.9 Non-selective moieties

In some embodiments, a compound of the invention is as shown in Table 2.

TABLE 2 Embodiments of compounds of the inventions. S. Variable groupsNo Prototype Structure R1 R2 1.

H, Me, Cl, CN, CF₃

2.

R₁ can be same H, F, Cl, OMe, Et 3.

R₁ can be same H, Me, Cl, CN, CF₃ 4.

R1 can be same R₂ H, Me R₃ H, F, Cl, OMe, Et 5.

R1 can be same H, Me, Cl 6.

R1 can be same — 7.

R1 can be same H, Me, Cl 8.

R1 can be same H, Me, Cl 9.

R1 can be same No R₂

In some embodiments, the compound of the invention is a compound shownin Table 3.

TABLE 3 Some exemplary compounds of the invention. CLASS SOLU- OF S.MOL. MOL. QUANT BILI- COM- NO. CODE STRUCTURE FOR. WT. (mg) TY POUND 1.SM 484A

C₁₇H₁₇Cl₂N₅ 362.26 5.7 DMSO AQ- pyrimidine 2. SM 484B

C₁₇H₁₇Cl₂N₅ 362.26 5.9 DMSO AQ- pyrimidine 3. SM 485

C₂₂H₂₇ClN₆ 410.94 4.5 DMSO AQ- pyrimidine 4. SM 488A

C₁₆H₃₅Cl₂N₅ 348.23 1.6 DMSO AQ- pyrimidine 5. SM 488B

C₁₆H₁₅Cl₂N₅ 348.23 3.5 DMSO AQ- pyrimidine 6. SM 489

C₂₀H₂₃ClN₆O 398.89 1.5 DMSO AQ- pyrimidine 7. SM 490

C₂₁H₂₅ClN₆ 396.92 3.8 DMSO AQ- pyrimidine 8. SM 491

C₂₁H₂₆ClN₇ 411.93 4.2 DMSO AQ- pyrimidine 9. SM 493

C₂₁H₂₅ClN₆O 412.92 3.7 DMSO AQ- pyrimidine 10. SM 494

C₂₂H₂₈ClN₇ 425.96 2.3 DMSO AQ- pyrimidine 11. SM 497

C₂₃H₃₀ClN₇ 439.98 1.7 DMSO AQ- pyrimidine 12. SM 498A

C₁₈H₁₉Cl₂N₅ 376.28 6.2 DMSO AQ- pyrimidine 13. SM 498B

C₁₈H₁₉Cl₂N₅ 376.28 8.7 DMSO AQ- pyrimidine 14. SM 499

C₂₃H₂₉ClN₆ 424.97 4.5 DMSO AQ- pyrimidine 15. SM 500

C₂₂H₂₇ClN₆O 426.94 4.6 DMSO AQ- pyrimidine 16. SM 501

C₂₃H₃₀ClN₇ 439.98 3.1 DMSO AQ- pyrimidine 17. SM 502

C₂₄H₃₂ClN₇ 454.01 3.8 DMSO AQ- pyrimidine 18. SM 504A

C₂₀H₂₃Cl₂N₅ 404.34 3.5 DMSO AQ- pyrimidine 19. SM 504B

C₂₀H₂₃Cl₂N₅ 404.34 2.5 DMSO AQ- pyrimidine 20. SM 505

C₂₅H₃₃ClN₆ 453.02 7.2 DMSO AQ- pyrimidine 21. SM 506

C₂₄H₃₁ClN₆O 455.00 3,1 DMSO AQ- pyrimidine 22. SM 507

C₂₅H₃₄ClN₇ 468.04 2.0 DMSO AQ- pyrimidine 23. SM 695A

C₁₅H₁₂Cl₃N₅ 368.65 7.6 DMSO AQ- pyrimidine 24. SM 695B

C₁₅H₁₂Cl₃N₅ 368.65 5.4 DMSO AQ- pyrimidine 25. SM 696

C₁₉H₂₀Cl₂N₆O 419.31 5.2 DMSO AQ- pyrimidine 26. SM 697

C₂₀H₂₂Cl₂N₆ 417.33 6.4 DMSO AQ- pyrimidine 27. SM 699

C₂₀H₂₂Cl₂N₆ 417.33 6.7 DMSO AQ- pyrimidine 28. SM 700

C₁₉H₂₀Cl₂N₆O 419.31 7.9 DMSO AQ- pyrimidine 29. SM 701

C₂₀H₂₃Cl₂N₇ 432.35 5.8 DMSO AQ- pyrimidine 30. SM 702

C₂₁H₂₅Cl₂N₇ 446.38 3.8 DMSO AQ- pyrimidine 31. SM 703

C₂₀H₂₃Cl₂N₇ 432.35 7.2 DMSO AQ- pyrimidine 32. SM 704

C₂₁H₂₅Cl₂N₇ 446.38 4.0 DMSO AQ- pyrimidine 33. SM 710A

C₁₆H₁₄Cl₃N₅ 382.67 6.7 DMSO AQ- pyrimidine 34. SM 710B

C₁₆H₁₄Cl₃N₅ 382.67 6.1 DMSO AQ- pyrimidine 35. SM 711

C₂₁H₂₄Cl₂N₆ 431.36 4.4 DMSO AQ- pyrimidine 36. SM 712

C₂₀H₂₂Cl₂N₆O 433.33 6.2 DMSO AQ- pyrimidine 37. SM 713

C₂₁H₂₅Cl₂N₇ 446.38 4.3 DMSO AQ- pyrimidine 38. SM 714

C₂₂H₂₇Cl₂N₇ 460.40 5.6 DMSO AQ- pyrimidine 39. SM 715

C₂₁H₂₄Cl₂N₆ 431.36 6.8 DMSO AQ- pyrimidine 40. SM 716

C₂₀H₂₂Cl₂N₆O 433.33 6.1 DMSO AQ- pyrimidine 41. SM 717

C₂₁H₂₅Cl₂N₇ 446.38 2.5 DMSO AQ- pyrimidine 42. SM 718

C₂₂H₂₇Cl₂N₇ 460.40 3.5 DMSO AQ- pyrimidine 43. SM 719A

C₁₆H₁₅Cl₂N₅ 348.23 5.6 DMSO AQ- pyrimidine 44. SM 719B

C₁₆H₁₅Cl₂N₅ 348.23 8.0 DMSO AQ- pyrimidine 45. SM 720

C₂₁H₂₅ClN₆ 396.92 5.2 DMSO AQ- pyrimidine 46. SM 721

C₂₀H₂₃ClN₆O 398.89 5.8 DMSO AQ- pyrimidine 47. SM 722

C₂₁H₂₆ClN₇ 411.93 1.3 DMSO AQ- pyrimidine 48. SM 723

C₂₂H₂₈ClN₇ 425.96 4.1 DMSO AQ- pyrimidine 49. SM 724

C₂₁H₂₅ClN₆ 396.92 5.0 DMSO AQ- pyrimidine 50. SM 725

C₂₀H₂₃ClN₆O 398.89 6.7 DMSO AQ- pyrimidine 51. SM 726

C₂₁H₂₆ClN₇ 411.93 5.7 DMSO AQ- pyrimidine 52. SM 727

C₂₂H₂₈ClN₇ 425.96 7.7 DMSO AQ- pyrimidine 53. SM 728A

C₁₅H₁₃Cl₂N₅ 334.20 7.2 DMSO AQ- pyrimidine 54. SM 728B

C₁₅H₁₃Cl₂N₅ 334.20 5.8 DMSO AQ- pyrimidine 55. SM 730

C₂₀H₂₃ClN₆ 382.89 6.6 DMSO AQ- pyrimidine 56. SM 731

C₁₉H₂₁ClN₆O 384.86 4.4 DMSO AQ- pyrimidine 57. SM 732

C₂₀H₂₃ClN₆ 382.89 4.1 DMSO AQ- pyrimidine 58. SM 733

C₁₉H₂₁ClN₆O 384.86 1.0 DMSO AQ- pyrimidine 59 SM 734

C₂₀H₂₄ClN₇ 397.90 3.2 DMSO AQ- pyrimidine 60. SM 332

C₂₅H₂₇ClN₈O₂ 506.99 1.5 DMSO AQ- triazine 61. SM 334

C₂₄H₂₄ClFN₈O 494.95 1.5 DMSO AQ- triazine 62. SM 335

C₂₆H₂₉ClN₈O 505.01 3.2 DMSO AQ- triazine 63. SM 336

C₂₄H₂₅ClN₈O 476.96 4.3 DMSO AQ- triazine 64. SM 337

C₂₆H₂₉ClN₈O2 521.01 8.1 DMSO AQ- triazine 65. SM 339

C₂₅H₂₆ClFN₈O 508.98 3.8 DMSO AQ- triazine 66. SM 340

C₂₇H₃₁ClN₈O 519.04 3.3 DMSO AQ- triazine 67. SM 341

C₂₅H₂₇ClN₈O 490.99 3.3 DMSO AQ- triazine 68. SM 345

C₂₂H₂₉ClN₈O₂ 472.97 1.7 DMSO AQ- triazine 69. SM 349

C₂₃H₃₁ClN₈O₂ 487.00 3.7 DMSO AQ- triazine 70. SM 350

C₂₁H₂₇ClN₈O₂ 458.94 5.7 DMSO AQ- triazine 71. SM 351

C₂₂H₂₉ClN₈O₂ 472.97 3.2 DMSO AQ- triazine 72. SM 352

C₂₀H₂₅ClN₈O₂ 444.92 3.7 DMSO AQ- triazine 73. SM 353

C₂₁H₂₇ClN₈O₂ 458.94 3.2 DMSO AQ- triazine 74. SM 356

C₂₇H₃₁ClN₈O₂ 535.04 3.7 DMSO AQ- triazine 75. SM 357

C₂₆H₂₈ClFN₈O 523.00 3.1 DMSO AQ- triazine 76 SM 358

C₂₈H₃₃ClN₈O 533.07 3.5 DMSO AQ- triazine 77. SM 359

C₂₆H₂₉ClN₈O 505.01 4.0 DMSO AQ- triazine 78. SM 360

C₂₂H₂₉ClN₈O₂ 472.97 5.3 DMSO AQ- triazine 79. SM 361

C₂₃H₃₁ClN₈O₂ 487.00 5.3 DMSO AQ- triazine 80. SM 362

C₂₄H₃₃ClN₈O₂ 501.02 4.0 DMSO AQ- triazine 81. SM 365

C₂₁H₂₇ClN₈O 442.95 4.8 DMSO AQ- triazine 82. SM 367

C₂₃H₃₁ClN₈O 471.00 3.0 DMSO AQ- triazine 83. SM 370

C₂₂H₂₉ClN₈O 456.97 5.7 DMSO AQ- triazine 84. SM 372

C₂₄H₃₃ClN₈O 485.02 1.5 DMSO AQ- triazine 85. SM 373

C₂₃H₃₁ClN₈O 471.00 3.8 DMSO AQ- triazine 86. SM 375

C₂₅H₃₅ClN₈O 499.05 4.5 DMSO AQ- triazine 87. SM 129

C₂₆H₂₃ClN₈ 482.97 1.7 DMSO AQ- triazine 88. SM 130

C₂₇H₃₁ClN₈O₃ 551.04 11.0 DMSO AQ- triazine 89. SM 132

C₂₈H₃₃ClN₈O₃ 567.07 12.1 DMSO AQ- triazine 90. SM 133

C₂₅H₂₂Cl₃N₉ 554.86 2.7 DMSO AQ- triazine 91. SM 135

C₂₀H₂₃Cl₂N₇O 448.35 3.1 DMSO AQ- triazine 92. SM 136

C₂₀H₂₇Cl₂N₇ 426.30 2.0 DMSO AQ- triazine 93. SM 137

C₁₈H₂₁Cl₂N₇ 406.31 2.2 DMSO AQ- triazine 94. SM 140

C₃₂H₄₁ClN₈O₃ 622.17 5.5 DMSO AQ- triazine 95. SM 142

C₂₂H₂₇ClN₈O₂ 470.96 2.5 DMSO AQ- triazine 96. SM 143

C₂₃H₂₉ClN₈O₂ 484.98 2.3 DMSO AQ- triazine 97. SM 144

C₂₈H₂₇ClN₈ 511.02 4.4 DMSO AQ- triazine 98. SM 146

C₂₉H₂₉ClN₈ 525.05 3.5 DMSO AQ- triazine 99. SM 148

C₃₂H₃₅ClN₈ 567.13 4.1 DMSO AQ- triazine 100. SM 151

C₂₉H₂₉ClN₈ 525.05 7.4 DMSO AQ- triazine 101. SM 189

C₂₆H₃₅ClN₈ 495.06 2.1 DMSO AQ- triazine 102. SM 190

C₂₇H₃₇ClN₈ 509.09 8.0 DMSO AQ- triazine 103. ATH 158

C₃₂H₃₅ClN₈O₃ 615.13 5.0 DMSO ACR- triazine 104. ATH 159

C₃₃H₃₇ClN₈O₃ 629.15 5.0 DMSO ACR- triazine 105. ATH 164

C₃₆H₃₆ClN₉O₂ 662.18 5.0 DMSO ACR- triazine 106. ATH 165

C₂₈H₂₉ClN₈O₂ 545.04 5.0 DMSO ACR- triazine 107. ATH 166

C₂₉H₃₁ClN₈O₂ 559.06 5.0 DMSO ACR- triazine 108 ATH 170

C₃₇H₃₈ClN₉O₃ 692.21 5.0 DMSO ACR- triazine 109. ATH 171

C₃₈H₄₀ClN₉O₃ 706.24 5.0 DMSO ACR- triazine 110. ATH 178

C₃₉H₄₁ClN₁₀O 701.26 5.0 DMSO ACR- triazine 111. ATH 179

C₄₀H₄₃ClN₁₀O 715.29 5.0 DMSO ACR- triazine 112. ATH 182

C₃₀H₃₁ClN₈O₃ 587.07 5.0 DMSO ACR- triazine 113. ATH 184

C₃₁H₃₃ClN₈O₃ 601.10 5.0 DMSO ACR- triazine 114. ATH 189

C₃₁H₃₃ClN₈O₂ 585.10 5.0 DMSO ACR- triazine 115. ATH 191

C₃₂H₃₅ClN₈O₂ 599.13 5.0 DMSO ACR- triazine 116. ATH 192

C₃₃H₃₇ClN₈O₃ 629.15 5.0 DMSO ACR- triazine 117. ATH 194

C₃₄H₃₉ClN₈O₃ 643.18 5.0 DMSO ACR- triazine 118. ATH 195

C₂₈H₂₉ClN₈O₂ 545.04 5.0 DMSO ACR- triazine 119. ATH 196

C₂₉H₃₁ClN₈O₂ 559.06 5.0 DMSO ACR- triazine 120. ATH 198

C₃₇H₃₈ClN₉O₂ 676.21 5.0 DMSO ACR- triazine 121. ATH 200

C₃₂H₃₅ClN₈O₂ 599.13 5.0 DMSO ACR- triazine 122. ATH 201

C₃₃H₃₇ClN₈O₂ 613.15 5.0 DMSO ACR- triazine 123. ATH 214

C₂₅H₂₅ClN₆O₂ 476.96 8.7 DMSO AQ- pyrimidine 124. ATH 218

C₁₇H₁₅Cl₂N₅ 360.24 7.0 DMSO AQ- pyrimidine 125. ATH 220

C₂₄H₂₃ClN₆O 446.93 8.2 DMSO AQ- pyrimidine 126. ATH 223

C₂₃H₂₀ClFN₆ 434.90 11.9 DMSO AQ- pyrimidine 127. ATH 226

C₂₃H₂₀BrClN₆ 495.80 7.2 DMSO AQ- pyrimidine 128. ATH 227

C₂₃H₂₀Cl₂N₆ 451.35 7.1 DMSO AQ- pyrimidine 129. ATH 228

C₂₁H₂₃ClN₆O 410.90 7.5 DMSO AQ- pyrimidine 130. ATH 231

C₂₂H₂₅ClN₆ 408.93 7.9 DMSO AQ- pyrimidine 131. ATH 232

C₂₃H₂₁ClN₆ 416.91 9.0 DMSO AQ- pyrimidine 132. ATH 236

C₂₃H₂₇ClN₆ 422.95 7.8 DMSO AQ- pyrimidine 133. ATH 237

C₁₈H₁₇Cl₂N₅ 374.27 8.5 DMSO AQ- pyrimidine 134. ATH 239

C₂₂H₂₅ClN₆O 424.93 6.1 DMSO AQ- pyrimidine 135. ATH 240

C₂₃H₂₇ClN₆ 422.95 7.9 DMSO AQ- pyrimidine 136. ATH 241

C₂₄H₂₉ClN₆ 436.98 7.4 DMSO AQ- pyrimidine 137. ATH 244

C₂₆H₂₇ClN₆O₂ 490.98 7.2 DMSO AQ- pyrimidine 138. ATH 245

C₂₅H₂₅ClN₆O 460.96 7.1 DMSO AQ- pyrimidine 139. ATH 247

C₂₄H₂₂ClFN₆ 448.92 6.2 DMSO AQ- pyrimidine 140. ATH 252

C₂₁H₂₃ClN₆ 394.90 9.0 DMSO AQ- pyrimidine 141. ATH 254

C₁₉H₂₁ClN6O 384.86 7.3 DMSO AQ- pyrimidine 142. ATH 255

C₂₀H₂₃ClN₆O 398.89 8.3 DMSO AQ- pyrimidine 143. ATH 256

C₂₂H₂₅ClN₆ 408.93 8.8 DMSO AQ- pyrimidine 144. ATH 257

C₁₉H₂₁ClN₆O 384.86 7.0 DMSO AQ- pyrimidine

In some embodiments, the compound of the invention is a compound shownin Table 4.

TABLE 4 Some exemplary compounds of the invention. S. No. Code StructureMol. formula Mol. Wt. Solubility Quantity 1 DKG805

C22H21ClN6 404.15162 DMSO 11.0 mg 2 DKG806

C23H23ClN6 418.16727 DMSO 11.2 mg 3 DKG807

C24H25ClN6 432.18292 DMSO 11.1 mg 4 DKG809

C21H18BrClN6 468.04648 DMSO 11.8 mg 5 DKG810

C22H20BrClN6 482.06213 DMSO 16.0 mg 6 DKG811

C23H22BrClN6 496.07778 DMSO 10.8 mg 7 DKG813

C21H18Cl2N6 424.09700 DMSO 11.0 mg 8 DKG814

C22H20Cl2N6 438.11265 DMSO 16.2 mg 9 DKG815

C23H22Cl2N6 452.12830 DMSO 16.8 mg 10 DKG817

C21H18ClFN6 408.12655 DMSO 14.5 mg 11 DKG818

C22H20ClFN6 422.14220 DMSO 16.0 mg 12 DKG819

C23H22ClFN6 436.15785 DMSO 15.6 mg 13 DKG821

C21H19ClN6 390.13597 DMSO 11.8 mg 14 DKG822

C22H21ClN6 404.15162 DMSO 11.0 mg 15 DKG823

C23H23ClN6 418.16727 DMSO 17.8 mg 16 DKG825

C22H21ClN6O 420.14654 DMSO 14.4 mg 17 DKG826

C23H23ClN6O 434.16219 DMSO 13.5 mg 18 DKG827

C24H25ClN6O 448.17784 DMSO 15.6 mg 19 DKG829

C23H23ClN6O2 450.15710 DMSO 20.8 mg 20 DKG830

C24H25ClN6O2 464.17275 DMSO 16.5 mg 21 DKG831

C25H27ClN6O2 478.18840 DMSO 19.2 mg 22 DKG833

C22H21ClN6 404.15162 DMSO 12.8 mg 23 DKG834

C23H23ClN6 418.16727 DMSO 15.2 mg 24 DKG835

C24H25ClN6 432.18292 13.5 mg 25 DKG837

C23H23ClN6O 434.16219 DMSO 17.6 mg 26 DKG838

C24H25ClN6O 448.17784 DMSO 12.5 mg 27 DKG839

C25H27ClN6O 462.19349 DMSO 12.1 mg 28 DKG845

C21H18Cl2N6 424.09700 DMSO 18.8 mg 29 DKG846

C23H22Cl2N6 452.12830 DMSO 17.6 mg 30 DKG847

C24H24Cl2N6 466.14395 DMSO 15.2 mg 31 DKG849

C22H20BrClN6 482.06213 DMSO 15.4 mg 32 DKG850

C23H22BrClN6 496.07778 DMSO 16.6 mg 33 DKG851

C24H24BrClN6 510.09343 DMSO 16.5 mg 34 DKG853

C22H20ClFN6 422.14220 DMSO 13.8 mg 35 DKG854

C23H22ClFN6 436.15785 DMSO 15.1 mg 36 DKG855

C24H24ClFN6 450.17350 DMSO 13.8 mg 37 DKG857

C23H23ClN6 418.16727 DMSO 13.1 mg 38 DKG858

C24H25ClN6 432.18292 DMSO 15.6 mg 39 DKG859

C25H27ClN6 446.19857 DMSO 14.5 mg

In some embodiments, the compound of the invention is a compound shownin Table 5.

TABLE 5 Some exemplary compounds of the invention. Com- S. pound- Mol.Formula, Amount No. ID IUPAC Name M.W. Solubility (mg) 40. MT1- 121

C19H23ClN6O, 386.88 MeOH, DMSO 10.4 41. MT1- 122

C20H25ClN6O, 400.91 MeOH, DMSO 12.0 42. MT1- 127

C18H21ClN6O, 372.85 MeOH, DMSO 12.9 43. MT1- 128

C21H27ClN6O, 414.93 MeOH, DMSO 13.8 44. MT1- 132

C22H29ClN6O, 428.96 MeOH, DMSO 10.7 45. MT1- 145

C19H23ClN6O, 386.88 MeOH, DMSO 10.7 46. MT1- 146

C20H25ClN6O, 400.91 MeOH, DMSO 10.5 47. MT1- 147

C21H27ClN6O, 414.93 MeOH, DMSO 13.0 48. MT1- 148

C22H29ClN6O, 428.96 MeOH, DMSO 10.3 49. MT1- 156

C19H23ClN6O, 386.88 MeOH, DMSO 12.4 50. MT1- 157

C20H25ClN6O, 400.91 MeOH, DMSO 11.0 51 MT1- 158

C21H27ClN6O, 414.93 MeOH, DMSO 10.9 52. MT1- 162

C22H29ClN6O, 428.96 MeOH, DMSO 14.0 53. MT1- 163

C23H31ClN6O, 442.98 MeOH, DMSO 14.1 54. MT1- 185

Cl8H21ClN6O, 372.85 MeOH, DMSO 12.1 55. MT1- 186

C19H23ClN6O, 386.88 MeOH, DMSO 12.3 56. MT1- 189

C20H25ClN6O, 400.91 MeOH, DMSO 11.3 57. MT1- 190

C21H27ClN6O, 414.93 MeOH, DMSO 10.6 58. MT1- 191

C22H29ClN6O 428.96 MeOH, DMSO 10.3 59. MT1- 192

C20H25ClN6O, 400.91 MeOH, DMSO 10.2 60. MT1- 193

C21H27ClN6O, 414.93 MeOH, DMSO 11.7 61. MT1- 194

C22H29ClN6O, 428.96 MeOH, DMSO 10.9 62. MT1- 195

C23H31ClN6O, 442.98 MeOH, DMSO 11.3 63. MT1- 196

C24H33ClN6O, 457.01 MeOH, DMSO 11.0

It should be recognized that the compounds described herein can bepresent and optionally administered in the form of salts, hydrates andprodrugs that are converted in vivo into the compounds described herein.For example, it is within the scope of the present invention to convertthe compounds described herein into and use them in the form of theirpharmaceutically acceptable salts derived from various organic andinorganic acids and bases in accordance with procedures well known inthe art.

When the compounds described herein possess a free base form, thecompounds can be prepared as a pharmaceutically acceptable acid additionsalt by reacting the free base form of the compound with apharmaceutically acceptable inorganic or organic acid, e.g.,hydrohalides such as hydrochloride, hydrobromide, hydroiodide; othermineral acids and their corresponding salts such as sulfate, nitrate,phosphate, etc.; and alkyl and monoarylsulfonates such asethanesulfonate, toluenesulfonate and benzenesulfonate; and otherorganic acids and their corresponding salts such as acetate, tartrate,maleate, succinate, citrate, benzoate, salicylate and ascorbate. Furtheracid addition salts amenable to the disclosed compounds include, but arenot limited to, adipate, alginate, arginate, aspartate, bisulfate,bisulfate, bromide, butyrate, camphorate, camphorsulfonate, caprylate,chloride, chlorobenzoate, cyclopentanepropionate, digluconate,dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate,galacterate (frommucic acid), galacturonate, glucoheptanoate, gluconate,glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate,hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, lactate,lactobionate, malate, malonate, mandelate, metaphosphate,methanesulfonate, methylbenzoate, monohydrogenphosphate,2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, pamoate,pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphate,phosphonate and phthalate. It should be recognized that the free baseforms will typically differ from their respective salt forms somewhat inphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free base forms for thepurposes of the present invention.

When the compounds described herein possess a free acid form, apharmaceutically acceptable base addition salt can be prepared byreacting the free acid form of the compound with a pharmaceuticallyacceptable inorganic or organic base. Examples of such bases are alkalimetal hydroxides including potassium, sodium and lithium hydroxides;alkaline earth metal hydroxides such as barium and calcium hydroxides;alkali metal alkoxides, e.g. potassium ethanolate and sodiumpropanolate; and various organic bases such as ammonium hydroxide,piperidine, diethanolamine and N-methylglutamine. Also included are thealuminum salts of the compounds of the present invention. Further basesalts amenable to the disclosed compounds include, but are not limitedto, copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium and zinc salts. Organic base salts include, but arenot limited to, salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, e.g., arginine, betaine,caffeine, chloroprocaine, choline, N,N′-dibenzylethylenediamine(benzathine), dicyclohexylamine, diethanolamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, iso-propylamine, lidocaine, lysine, meglumine,N-methyl-D-glucamine, morpholine, piperazine, piperidine, polyamineresins, procaine, purines, theobromine, triethanolamine, triethylamine,trimethylamine, tripropylamine andtris-(hydroxymethyl)-methylamine(tromethamine). It should be recognizedthat the free acid forms will typically differ from their respectivesalt forms somewhat in physical properties such as solubility in polarsolvents, but otherwise the salts are equivalent to their respectivefree acid forms for the purposes of the present invention.

Compounds of the present invention that comprise basicnitrogen-containing groups can be quaternized with such agents asC₁-C₄alkyl halides, e.g., methyl, ethyl, iso-propyl and tert-butylchlorides, bromides and iodides; di(C₁-C₄alkyl) sulfates, e.g.,dimethyl, diethyl and diamyl sulfates; C₁₀-C₁₈alkyl halides, e.g.,decyl, dodecyl, lauryl, myristyl and stearyl chlorides, bromides andiodides; and aryl(C₁-C₄alkyl) halides, e.g., benzyl chloride andphenethyl bromide. Such salts permit the preparation of bothwater-soluble and oil-soluble compounds described herein.

Prodrug derivatives of compounds described herein can be prepared bymodifying substituents of compounds described herein that are thenconverted in vivo to a different substituent. It is noted that in manyinstances, the prodrugs themselves also fall within the scope of therange of compounds described herein. For example, prodrugs can beprepared by reacting a compound with a carbamylating agent (e.g.,1,1-acyloxyalkylcarbonochloridate, para-nitrophenyl carbonate, or thelike) or an acylating agent. Further examples of methods of makingprodrugs are described in Saulnier et al., 1994, Bioorganic andMedicinal Chemistry Letters, Vol. 4, p. 1985.

Protected derivatives of compounds described herein can also be made.Examples of techniques applicable to the creation of protecting groupsand their removal can be found in P. G. M. Wuts and T. W. Greene,Greene's Protecting Groups in Organic Synthesis, 4^(th) edition, JohnWiley & Sons, Inc. 2006.

Compounds described herein can also be conveniently prepared or formedduring the process of the invention, as solvates (e.g. hydrates).Hydrates of the compounds described herein can be conveniently preparedby recrystallization from an aqueous/organic solvent mixture, usingorganic solvents such as dioxin, tetrahydrofuran or methanol.

In some embodiments, the compounds of the present disclosure can be usedin the form of a pharmaceutically acceptable salt. A “pharmaceuticallyacceptable salt”, as used herein, is intended to encompass any compounddescribed herein that is utilized in the form of a salt thereof,especially where the salt confers on the compound improvedpharmacokinetic properties as compared to the free form of compound or adifferent salt form of the compound. The pharmaceutically acceptablesalt form can also initially confer desirable pharmacokinetic propertieson the compound that it did not previously possess, and may evenpositively affect the pharmacodynamics of the compound with respect toits therapeutic activity in the body. An example of a pharmacokineticproperty that can be favorably affected is the manner in which thecompound is transported across cell membranes, which in turn maydirectly and positively affect the absorption, distribution,biotransformation and excretion of the compound. While the route ofadministration of the pharmaceutical composition is important, andvarious anatomical, physiological and pathological factors cancritically affect bioavailability, the solubility of the compound isusually dependent upon the character of the particular salt formthereof, which it utilized. One of skill in the art will appreciate thatan aqueous solution of the compound will provide the most rapidabsorption of the compound into the body of a subject being treated,while lipid solutions and suspensions, as well as solid dosage forms,will result in less rapid absorption of the compound.

Pharmaceutically acceptable salts include those derived from inorganicacids such as sulfuric, sulfamic, phosphoric, nitric, and the like; andthe salts prepared from organic acids such as acetic, propionic,succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.See, for example, Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19 (1977), the content of which is herein incorporated by referencein its entirety. Exemplary salts also include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,succinate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthalate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. Suitable acids which are capable offorming salts with the compounds of the disclosure include inorganicacids such as hydrochloric acid, hydrobromic acid, perchloric acid,nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and thelike; and organic acids such as 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid,3-phenylpropionic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylicacid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), acetic acid,anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonicacid, cinnamic acid, citric acid, cyclopentanepropionic acid,ethanesulfonic acid, formic acid, fumaric acid, glucoheptonic acid,gluconic acid, glutamic acid, glycolic acid, heptanoic acid,hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid,malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconicacid, naphthalene sulfonic acid, o-(4-hydroxybenzoyl)benzoic acid,oxalic acid, p-chlorobenzenesulfonic acid, propionic acid,p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid,succinic acid, sulfanilic acid, tartaric acid, tertiary butylaceticacid, trifluoroacetic acid, trimethylacetic acid, and the like. Suitablebases capable of forming salts with the compounds of the disclosureinclude inorganic bases such as sodium hydroxide, ammonium hydroxide,sodium carbonate, calcium hydroxide, potassium hydroxide and the like;and organic bases such as mono-, di- and tri-alkyl and aryl amines(e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine,N-methylglucamine, pyridine, picoline, dicyclohexylamine,N,N′-dibezylethylenediamine, and the like), and optionally substitutedethanol-amines (e.g., ethanolamine, diethanolamine, triethanolamine andthe like),

The compounds according to the disclosure can be prepared by syntheticprocesses which are known to those skilled in the art, particularly inview of the state of the art and the specific preparatory examplesprovided below herein. Suitable modification to starting materials bymethods well known in the art can also be employed. It is noted,however, that the compounds described herein can also be synthesized byother synthetic routes that others may devise.

It will be readily recognized that certain compounds described hereinhave atoms with linkages to other atoms that confer a particularstereochemistry to the compound (e.g., chiral centers). It is recognizedthat synthesis of compounds according to the present invention canresult in the creation of mixtures of different stereoisomers(enantiomers, diastereomers). Unless a particular stereochemistry isspecified, recitation of a compound is intended to encompass all of thedifferent possible stereoisomers.

Various methods for separating mixtures of different stereoisomers areknown in the art. For example, a racemic mixture of a compound may bereacted with an optically active resolving agent to form a pair ofdiastereoisomeric compounds. The diastereomers may then be separated inorder to recover the optically pure enantiomers. Dissociable complexesmay also be used to resolve enantiomers (e.g., crystallinediastereoisomeric salts). Diastereomers typically have sufficientlydistinct physical properties (e.g., melting points, boiling points,solubilities, reactivity, etc.) that they can be readily separated bytaking advantage of these dissimilarities. For example, diastereomerscan typically be separated by chromatography or by separation/resolutiontechniques based upon differences in solubility. A more detaileddescription of techniques that can be used to resolve stereoisomers ofcompounds from their racemic mixture can be found in Jean Jacques AndreCollet, Samuel H. Wilen, Enantiomers, Racemates and Resolutions, JohnWiley & Sons, Inc. (1981).

The compounds described herein showed significant in vitro/in vivoactivity against Plasmodium falciparum, without any toxicity.Accordingly, in one aspect, the compounds disclosed herein are usefulfor treatment of infectious diseases such as malarial or any otherdisease in question.

Inventors have discovered that the compounds described herein are alsounexpectedly, surprisingly agonists of orphan nuclear receptor Nurr1.Accordingly, the compounds described herein can be administered to asubject as part of a therapeutic application. In general, the method canbe characterized as including a step of administering a therapeuticallyeffective amount of the compound to a subject in need thereof.

In one embodiment, the compounds and method disclosed herein can be usedin treating a subject for a disease state in which a decreased Nurr1activity contributes to the pathology or symptomology of the disease.Disease states in which Nurr1 activity is decreased include, but are notlimited to, neurodegenerative diseases, inflammation diseases ordisorders, restless leg syndrome, and the like. The compounds andmethods disclosed herein can also be used in treating a subject for adisease for which a dopamine agonist is a useful treatment, for examplerestless leg syndrome.

The disclosure also provides a method for causing the differentiation ofa stem cell, e.g., a human embryonic stem cell, into a dopaminergicneuron by contacting the stem cell with a compound described herein thatis present in an amount sufficient to induce differentiation of the stemcell. Without limitation, the stem cell can be contacted with thecompounds described herein in a cell culture e.g., in vitro or ex vivo,or administrated to a subject, e.g., in vivo. In some embodiments of theinvention, a compound described herein can be administrated to a subjectto treat, prevent, and/or diagnose neurodegenerative disorders,including those described herein.

The term “contacting” or “contact” as used herein in connection withcontacting a stem cell includes subjecting the cell to an appropriateculture media which comprises the indicated compound. Where the stemcell is in vivo, “contacting” or “contact” includes administering thecompound in a pharmaceutical composition to a subject via an appropriateadministration route such that the compound contacts the stem cell invivo.

By “an amount sufficient to induce differentiation” of a stem cell ismeant the amount of a compound of the invention required to cause anundifferentiated stem cell to differentiate into a desired cell type,e.g., a neuron.

By “differentiation” is meant the process whereby an unspecialized stemcell acquires the features of a specialized cell, e.g., a nerve cell.Differentiation can also refer to the restriction of the potential of acell to self-renew and is generally associated with a change in thefunctional capacity of the cell. Differentiation of a stem cell can bedetermined by methods well known in the art, including analysis for cellmarkers or morphological features associated with cells of a defineddifferentiated state. Examples of such markers and features includemeasurement of glycoprotein, alkaline phosphatase, and carcinoembryonicantigen expression, where an increase in any one of these proteins is anindicator of differentiation.

By “stem cell” is meant any cell with the potential to self-renew and,under appropriate conditions, differentiate into a dedicated progenitorcell or a specified cell or tissue. Stem cells can be pluripotent ormultipotent. Stem cells include, but are not limited to embryonic stemcells, embryonic germ cells, adult stem cells, and umbilical cord bloodcells.

Stem cells are unique cell populations that have the ability to divide(self-renew) for indefinite periods of time, and, under the rightconditions or signals, to differentiate into the many different celltypes that make up an organism. Stem cells derived from the inner cellmass of the blastocyst are known as embryonic stem (ES) cells. Stemcells derived from the primordial germ cells, and which normally developinto mature gametes (eggs and sperm) are known as embryonic germ (EG)cells. Both of these types of stem cells are known as pluripotent cellsbecause of their unique ability to differentiate into derivatives of allthree embryonic germ layers (endoderm, mesoderm, and ectoderm).

The pluripotent stem cells can further specialize into another type ofmultipotent stem cell often derived from adult tissues. Multipotent stemcells are also able to undergo self renewal and differentiation, butunlike embryonic stem cells, are committed to give rise to cells thathave a particular function. Examples of adult stem cells includehematopoietic stem cells (HSC), which can proliferate and differentiateto produce lymphoid and myeloid cell types; bone marrow derived stemcells (BMSC), which can differentiate into adipocytes, chondrocytes,osteocytes, hepatocytes, cardiomyocytes and neurons; neural stem cells(NSC), which can differentiate into astrocytes, neurons, andoligodendrocytes; and peripheral blood stem cells. Multipotent stemcells have also been derived from epithelial and adipose tissues andumbilical cord blood (UCB).

ES cells, derived from the inner cell mass of preimplantation embryos,have been recognized as the most pluripotent stem cell population andare therefore the preferred cell for the methods of the invention. Thesecells are capable of unlimited proliferation ex vivo, while maintainingthe capacity for differentiation into a Wide variety of somatic andextra-embryonic tissues. ES cells can be male Q(Y) or female (XX);female ES cells are preferred.

Multipotent, adult stem cells can also be used in the methods of theinvention. Preferred adult stem cells include hematopoietic stem cells(HSC), Which can proliferate and differentiate throughout life toproduce lymphoid and myeloid cell types; bone marrow-derived stem cells(BMSC), Which can differentiate into various cell types includingadipocytes, chondrocytes, osteocytes, hepatocytes, cardiomyocytes andneurons; and neural stem cells (NSC), Which can differentiate intoastrocytes, neurons, and oligodendrocytes. Multipotent stem cellsderived from epithelial and adipose tissues and umbilical cord bloodcells can also be used in the methods of the invention.

Stem cells can be derived from source, e.g., any mammal including, butnot limited to, mouse, human, and primates. Following acquisition ofstem cells, these cells can be used directly in the methods disclosedherein. For example, umbilical cord blood cells can be acquired insufficient quantity to use directly for therapeutic purposes.Alternatively, stem cells can first be expanded in order to increase thenumber of available cells. See, for example, U.S. Pat. No. 6,338,942,content of which is incorporated herein by reference in its entirety.Exemplary mouse strains for stem cell preparation include 129, C57BL/6,and a hybrid strain (Brook et al., Proc. Natl. Acad. Sci. U.S.A.94:5709-5712 (1997), Baharvand et al., In Vitro Cell Dev. Biol. Anim.40:76-81 (2004), content of both of which is incorporated herein byreference). Methods for preparing mouse, human, or primate stem cellsare known in the art and are described, for example, in Nagy et al.,Manipulating the mouse embryo: A laboratory manual, 3rd ed., Cold SpringHarbor Laboratory Press (2002); Thomson et al., Science 282:1145-1147(1998), Marshall et al., Methods Mol. Biol. 158: 1 1-18 (2001); Thomsonet al., Trends Biotechnol. 18:5357 (2000); Jones et al., Semin. Reprod.Med. 18:219-223 (2000); Voss et al., Exp. Cell Res. 230:45-49 (1997);and Odorico et al., Stem Cells 19:193-204 (2001), content of all whichis incorporated herein by reference in its entirety.

ES cells can be directly derived from the blastocyst or any other earlystage of development, or can be a “cloned” stem cell line derived fromsomatic nuclear transfer and other similar procedures. General methodsfor culturing mouse, human, or primate ES cells from a blastocyst can befound in Appendix C of the NIH report on stem cells entitled Stem Cells:Scientific Progress and Future Research Directions (June 2001), contentof which is incorporated herein by reference. For example, in the firststep, the inner cell mass of a preimplantation blastocyst is removedfrom the trophectoderm that surrounds it. (For cultures of human EScells, blastocysts are generated by in vitro fertilization and donatedfor research.) The small plastic culture dishes used to grow the cellscontain growth medium supplemented with fetal calf serum, and aresometimes coated with a “feeder” layer of nondividing cells. The feedercells are often mouse embryonic fibroblast (MEF) cells that have beenchemically inactivated so they will not divide. Additional reagents,such as the cytokine leukemia inhibitory factor (LIF), can also be addedto the culture medium for mouse ES cells. Second, after several days toa week, proliferating colonies of cells are removed and dispersed intonew culture dishes, each of which may or may not contain an MEF feederlayer. If the cells are to be used to human therapeutic purposes, it ispreferable that the MEF feeder layer is not included. Under these exvivo conditions, the ES cells aggregate to form colonies. In the thirdmajor step required to generate ES cell lines, the individual,nondifferentiating colonies are dissociated and replated into newdishes, a step called passage. This replating process establishes a“line” of ES cells. The line of cells is termed “clonal” if a single EScell generates it. Limiting dilution methods can be used to generate aclonal ES cell line. Reagents needed for the culture of stem cells arecommercially available, for example, from Invitrogen, Stem CellTechnologies, R&D Systems, and Sigma Aldrich, and are described, forexample, in U.S. Patent Publication Nos. 2004/0235159 and 2005/0037492and Appendix C of the NIH report, Stem Cells: Scientific Progress andFuture Research Directions, supra.

For in vivo methods, a therapeutically effective amount of a compounddescribed herein can be administered to a subject. Methods ofadministering compounds to a subject are known in the art and easilyavailable to one of skill in the art.

As one of skill in the art is aware, promoting stem cell differentiationin a subject can lead to treatment, prevention or amelioration of anumber of neurodegenerative disorders. By “neurodegenerative disorder”is meant any disease or disorder caused by or associated with thedeterioration of cells or tissues of the nervous system. Exemplaryneurodegenerative disorders are polyglutamine expansion disorders (e.g.,HD, dentatorubropallidoluysian atrophy, Kennedy's disease (also referredto as spinobulbar muscular atrophy), and spinocerebellar ataxia (e.g.,type 1, type 2, type 3 (also referred to as Machado-Joseph disease),type 6, type 7, and type 17)), other trinucleotide repeat expansiondisorders (e.g., fragile X syndrome, fragile XE mental retardation,Friedreich's ataxia, myotonic dystrophy, spinocerebellar ataxia type 8,and spinocerebellar ataxia type 12), Alexander disease, Alper's disease,Alzheimer disease, amyotrophic lateral sclerosis (ALS), ataxiatelangiectasia, Batten disease (also referred to asSpielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockaynesyndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, ischemiastroke, Krabbe disease, Lewy body dementia, multiple sclerosis, multiplesystem atrophy, Parkinson's Disease, Pelizaeus-Merzbacher disease,Pick's disease, primary lateral sclerosis, Refsum's disease, Sandhoffdisease, Schilder's disease, spinal cord injury, spinal muscular atrophy(SMA), SteeleRichardson-Olszewski disease, and Tabes dorsalis.

In some embodiments, the neurodegenerative disease or disorder isParkinson's Disease.

Methods of diagnosing subjects as having or being at risk of havingParkinson's Disease are well-known in the art. For example, the presenceof one or more of the following symptoms can be used as part of a PDdiagnosis: trembling, e.g., an involuntary, rhythmic tremor of one armor one leg; muscular rigidity, stiffness, or discomfort; generalslowness in any of the activities of daily living, e.g., akinesia orbradykinesia; difficulty with walking, balance, or posture; alterationin handwriting; emotional changes; memory loss; speech problems; anddifficulty sleeping. Review of a subject's symptoms, activity,medications, concurrent medical problems, or possible toxic exposurescan be useful in making a PD diagnosis. In addition, a subject can betested for the presence or absence of genetic mutations that canindicate an increased likelihood of having Parkinson's Disease. Forexample, the presence of one or more specific mutations or polymorphismsin the NURR1, alpha-synuclein, parkin, MAPT, DJ-1, PINK1, SNCA, NAT2, orLRRK2 genes can be used to diagnose a subject as having or being at riskof having Parkinson's Disease. See, e.g., U.S. Patent ApplicationPublication Nos. 2003-0119026 and 2005-0186591; Bonifati, Minerva Med.96:175-186, 2005; and Cookson et al., Curr. Opin. Neurol. 18:706-711,2005, content of each of which is incorporated herein by reference.

Without limitation, the methods for utilizing stem cells describedherein can be used for the treatment of diseases treatable throughtransplantation of differentiated cells derived from ES cells. Stemcells of the invention or produced using the methods disclosed hereincan be used to treat neurodegenerative diseases, such as Parkinson'sdisease, Alzheimer's disease, or traumatic injury to the brain or spinalcord in any subject, e.g. a human.

The inventor have also discovered that the compounds disclosed hereinalso inhibit or reduce the expression of pro-inflammatory cytokine genesin primary microglia derived from P1 rat brains. Accordingly, thecompounds disclosed herein can also be used for treating diseases ordisorders characterized by elevated levels pro-inflammatory mediatorsand/or elevated levels of pro-inflammatory mediator gene expression.Accordingly, in another aspect the disclosure provides a method fortreating a subject suffering from a disease or disorder characterized byelevated levels pro-inflammatory mediators and/or elevated levels ofpro-inflammatory mediator gene expression, the method comprisingadministering an effective amount of a compound disclosed herein to thesubject. Exemplary pro-inflammatory mediators include, but are notlimited to, pro-inflammatory cytokines, leukocytes, leukotiens,prostaglandins and other mediators involved in the initiation andmaintenance of inflammation. Pro-inflammatory cytokines and inflammationmediators include, but are not limited to, IL-1-alpha, IL-1-beta, IL-6,IL-8, IL-11, IL-12, IL-17, IL-18, TNF-alpha, leukocyte inhibitory factor(LIF), IFN-gamma, Oncostatin M (OSM), ciliary neurotrophic factor(CNTF), TGF-beta, granulocyte-macrophage colony stimulating factor(GM-CSF), iNOS, and chemokines that chemoattract inflammatory cells. Anumber of assays for in vivo state of inflammation are known in the artwhich can be utilized for measuring pro-inflammatory mediator levels.See for example U.S. Pat. Nos. 5,108,899 and 5,550,139, contents of bothof which are herein incorporated by reference.

In some embodiments of the aspects described herein, the disease,disorder, or disease condition characterized by elevated levelspro-inflammatory cytokines and/or elevated levels of pro-inflammatorycytokine gene expression is an autoimmune disease, neurodegenerativedisease, inflammation, an inflammation associated disorder, a diseasecharacterized by inflammation, or a pathogen or non-pathogen infection.

As used herein, the term “autoimmune disease” refers to disease ordisorders wherein the immune system of a subject, e.g., a mammal, mountsa humoral or cellular immune response to the subject's own tissue or toantigenic agents that are not intrinsically harmful to the subject,thereby producing tissue injury in such a subject. Examples of suchdisorders include, but are not limited to, systemic lupus erythematosus(SLE), mixed connective tissue disease, scleroderma, Sjögren's syndron,rheumatoid arthritis, and Type I diabetes.

As used herein, the term “neurodegenerative disease or disorder”includes any disease disorder or condition that affects neuronalhomeostasis, e.g., results in the degeneration or loss of neuronalcells. Neurodegenerative diseases include conditions in which thedevelopment of the neurons, i.e., motor or brain neurons, is abnormal,as well as conditions in which result in loss of normal neuron function.Examples of such neurodegenerative disorders include Alzheimer's diseaseand other tauopathies such as frontotemporal dementia, frontotemporaldementia with Parkinsonism, frontotemporal lobe dementia,pallidopontonigral degeneration, progressive supranuclear palsy,multiple system tauopathy, multiple system tauopathy with preseniledementia, Wilhelmsen-Lynch disease,disinhibition-dementia-park-insonism-amytrophy complex, Pick's disease,or Pick's disease-like dementia, corticobasal degeneration, frontaltemporal dementia, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis, Friedreich'sataxia, Lewybody disease, spinal muscular atrophy, and parkinsonismlinked to chromosome 17.

As used herein, the term “inflammation” refers to any cellular processesthat lead to the activation of caspase-1, or caspase-5, the productionof cytokines IL-I, IL-6, IL-8, TNF-alpha, iNOS, and/or the relateddownstream cellular events resulting from the actions of the cytokinesthus produced, for example, fever, fluid accumulation, swelling, abscessformation, and cell death. As used herein, the term “inflammation”refers to both acute responses (i.e., responses in which theinflammatory processes are active) and chronic responses (i.e.,responses marked by slow progression and formation of new connectivetissue). Acute and chronic inflammation may be distinguished by the celltypes involved. Acute inflammation often involves polymorphonuclearneutrophils; whereas chronic inflammation is normally characterized by alymphohistiocytic and/or granulomatous response.

As used herein, the term “inflammation” includes reactions of both thespecific and non-specific defense systems. A specific defense systemreaction is a specific immune system reaction response to an antigen(possibly including an autoantigen). A non-specific defense systemreaction is an inflammatory response mediated by leukocytes incapable ofimmunological memory. Such cells include granulocytes, macrophages,neutrophils and eosinophils. Examples of specific types of inflammationinclude, but are not limited to, diffuse inflammation, focalinflammation, croupous inflammation, interstitial inflammation,obliterative inflammation, parenchymatous inflammation, reactiveinflammation, specific inflammation, toxic inflammation and traumaticinflammation.

As used herein, the term “pathogen infection” refers to infection with apathogen. As used herein the term “pathogen” refers to an organism,including a microorganism, which causes disease in another organism(e.g., animals and plants) by directly infecting the other organism, orby producing agents that causes disease in another organism (e.g.,bacteria that produce pathogenic toxins and the like). As used herein,pathogens include, but are not limited to bacteria, protozoa, fungi,nematodes, viroids and viruses, or any combination thereof, wherein eachpathogen is capable, either by itself or in concert with anotherpathogen, of eliciting disease in vertebrates including but not limitedto mammals, and including but not limited to humans. As used herein, theterm “pathogen” also encompasses microorganisms which may not ordinarilybe pathogenic in a non-immunocompromised host. Specific nonlimitingexamples of viral pathogens include Herpes simplex virus (HSV)1, HSV2,Epstein Barr virus (EBV), cytomegalovirus (CMV), human Herpes virus(HHV) 6, HHV7, HHV8, Varicella zoster virus (VZV), hepatitis C,hepatitis B, HIV, adenovirus, Eastern Equine Encephalitis Virus (EEEV),West Nile virus (WINE), JC virus (JCV) and BK virus (BKV).

As used herein, the term “microorganism” includes prokaryotic andeukaryotic microbial species from the Domains of Archaea, Bacteria andEucarya, the latter including yeast and filamentous fungi, protozoa,algae, or higher Protista. The terms “microbial cells” and “microbes”are used interchangeably with the term microorganism.

As used herein, the term “bacteria,” or “bubacteria”, refers to a domainof prokaryotic organisms. Bacteria include at least 11 distinct groupsas follows: (1) Gram-positive (gram+) bacteria, of which there are twomajor subdivisions: (i) high G+C group (Actinomycetes, Mycobacteria,Micrococcus, others) (ii) low G+C group (Bacillus, Clostridia,Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram-positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of Grampositive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

As used herein, the term “specific defense system” is intended to referto that component of the immune system that reacts to the presence ofspecific antigens. Inflammation is said to result from a response of thespecific defense system if the inflammation is caused by, mediated by,or associated with a reaction of the specific defense system. Examplesof inflammation resulting from a response of the specific defense systeminclude the response to antigens such as rubella virus, autoimmunediseases such as lupus erythematosus, rheumatoid arthritis, Reynaud'ssyndrome, multiple sclerosis etc., delayed type hypersensitivityresponse mediated by T-cells, etc. Chronic inflammatory diseases and therejection of transplanted tissue and organs are further examples ofinflammatory reactions of the specific defense system.

As used herein, a reaction of the “non-specific defense system” isintended to refer to a reaction mediated by leukocytes incapable ofimmunological memory. Such cells include granulocytes and macrophages.As used herein, inflammation is said to result from a response of thenonspecific defense system, if the inflammation is caused by, mediatedby, or associated with a reaction of the non-specific defense system.Examples of inflammation which result, at least in part, from a reactionof the non-specific defense system include inflammation associated withconditions such as: adult respiratory distress syndrome (ARDS) ormultiple organ injury syndromes secondary to septicemia or trauma;reperfusion injury of myocardial or other tissues; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders; thermal injury; hemodialysis;leukophoresis; ulcerative colitis; Crohn's disease; necrotizingenterocolitis; granulocyte transfusion associated syndromes; andcytokine-induced toxicity. The term immune-mediated refers to a processthat is either autoimmune or inflammatory in nature.

In some embodiments of the aspects described herein, theinflammation-associated disorder or disease characterized byinflammation is selected from the group consisting of asthma, autoimmunediseases, chronic prostatitis, glomerulonephritis, inflammatory bowldisease, pelvic inflammatory disease, reperfusion injury, arthritis,silicosis, vasculitis, inflammatory myopathies, hypersensitivities,migraine, psoriasis, gout, atherosclerosis, and any combinationsthereof.

Exemplary inflammatory diseases include, but are not limited to,rheumatoid arthritis, inflammatory bowel disease, pelvic inflammatorydisease, ulcerative colitis, psoriasis, systemic lupus erythematosus,multiple sclerosis, type 1 diabetes mellitus, multiple sclerosis,psoriasis, vasculitis, and allergic inflammation such as allergicasthma, atopic dermatitis, and contact hypersensitivity. Other examplesof auto-immune-related diseases or disorders, include but should not beconstrued to be limited to, rheumatoid arthritis, multiple sclerosis(MS), systemic lupus erythematosus, Graves' disease (overactivethyroid), Hashimoto's thyroiditis (underactive thyroid), Type 1 diabetesmellitus, celiac disease, Crohn's disease and ulcerative colitis,Guillain-Barre syndrome, primary biliary sclerosis/cirrhosis, sclerosingcholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma,Sjogren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis,polymyalgia rheumatica, temporal arteritis/giant cell arteritis, chronicfatigue syndrome CFS), psoriasis, autoimmune Addison's Disease,ankylosing spondylitis, Acute disseminated encephalomyelitis,antiphospholipid antibody syndrome, aplastic anemia, idiopathicthrombocytopenic purpura, Myasthenia gravis, opsoclonus myoclonussyndrome, optic neuritis, Ord's thyroiditis, pemphigus, perniciousanaemia, polyarthritis in dogs, Reiter's syndrome, Takayasu's arteritis,warm autoimmune hemolytic anemia, Wegener's granulomatosis, fibromyalgia(FM), autoinflammatory PAPA syndrome, Familial Mediterranean Fever,familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and theneonatal onset multisystem inflammatory disease.

As used herein, an anti-inflammation treatment aims to prevent or slowdown (lessen) an undesired physiological change or disorder, such as thedevelopment or progression of the inflammation. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of inflammation diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. An anti-inflammation treatment can also mean prolongingsurvival as compared to expected survival if not receiving treatment. Ananti-inflammation treatment can also completely suppress theinflammation response.

One goal of anti-inflammatory treatment is to bring pro-inflammatorymediator levels down to as close to normal as is safely possible.Accordingly, in one embodiment, level of at least one pro-inflammatorymediator in the subject undergoing treatment is reduced by at least 5%,at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%relative to a reference level. A reference level can be the level of thepro-inflammatory mediator in the subject before onset of treatmentregime.

The compounds, compositions, and methods disclosed herein can also beused for research purposes for the study of differentiation ordevelopment, and for the generation of transgenic animals useful forresearch purposes. The stem cells and methods of their use describedherein can be used, for example, to create and test animal models ofParkinson's Disease or other neurological disorders. The stem cells andmethods of the invention can also be used to study the effects of aparticular compound on stem cell differentiation, development, andtissue generation or regeneration.

For administration to a subject, the compounds described herein can beprovided in pharmaceutically acceptable (e.g., sterile) compositions.Accordingly, another aspect described herein is a pharmaceuticalcomposition comprising a compound disclosed herein and apharmaceutically acceptable carrier. These pharmaceutically acceptablecompositions comprise an effective amount of one or more of thecompounds described herein, formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Asdescribed in detail below, the pharmaceutical compositions of thepresent disclosure can be specifically formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), lozenges, dragees, capsules, pills, tablets(e.g., those targeted for buccal, sublingual, and/or systemicabsorption), boluses, powders, granules, pastes for application to thetongue; (2) parenteral administration, for example, by subcutaneous,intramuscular, intravenous (e.g., bolus or infusion) or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8)transmucosally; or (9) nasally. Additionally, compounds can be implantedinto a patient or injected using a drug delivery system. See, forexample, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236(1984); Lewis, ed. “Controlled Release of Pesticides andPharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. Nos.3,773,919; and 35 3,270,960, content of all of which is hereinincorporated by reference.

As used herein, the term “pharmaceutically acceptable” or“pharmacologically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio. Moreover, for animal (e.g., human)administration, it will be understood that compositions should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, disintegrating agents, binders, sweetening agents, flavoringagents, perfuming agents, protease inhibitors, plasticizers,emulsifiers, stabilizing agents, viscosity increasing agents, filmforming agents, solubilizing agents, surfactants, preservative andantioxidants can also be present in the formulation. The terms such as“excipient”, “carrier”, “pharmaceutically acceptable carrier” or thelike are used interchangeably herein.

For liquid formulations, pharmaceutically acceptable carriers can beaqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions, orsuspensions, including saline and buffered media. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil. Solutions or suspensions can also include one or more ofthe following components: a sterile diluent, including water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol and other synthetic solvents; antibacterial agents,including benzyl alcohol and methyl parabens; antioxidants, includingascorbic acid or sodium bisulfate; chelating agents, includingethylenediaminetetraacetic acid (EDTA); buffers, including acetates,citrates and phosphates, and agents for the adjustment of tonicity,including sodium chloride and dextrose. The pH can be adjusted withacids or bases, including hydrochloric acid and sodium hydroxide.

Liposomes and non-aqueous vehicles such as fixed oils can also be used.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

As indicated above, the compositions can further comprise binders (e.g.,acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g., cornstarch, potato starch, alginic acid,silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodiumstarch glycolate, Primogel), buffers (e.g., tris-HCl, acetate,phosphate) of various pH and ionic strength, additives such as albuminor gelatin to prevent absorption to surfaces, detergents (e.g., Tween20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors,surfactants (e.g., sodium lauryl sulfate), permeation enhancers,solubilizing agents (e.g., glycerol, polyethylene glycerol), a glidant(e.g., colloidal silicon dioxide), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.,hydroxypropyl cellulose, hydroxypropylmethyl cellulose), viscosityincreasing agents (e.g., carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citricacid), flavoring agents (e.g., peppermint, methyl salicylate, or orangeflavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens),lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol,sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide),plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers(e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate),polymer coatings (e.g., poloxamers or poloxamines), coating and filmforming agents (e.g., ethyl cellulose, acrylates, polymethacrylates)and/or adjuvants.

It is especially advantageous to formulate oral and intravenouscompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals. The pharmaceutical compositions can beincluded in a container, pack, or dispenser together with instructionsfor administration.

The amount of a compound described herein that can be combined with acarrier material to produce a single dosage form will generally be aneffective amount of the compound. A pharmaceutical composition typicallycontains an amount of at least 0.01 weight % of active ingredient, i.e.,a compound of this disclosure, per weight of total pharmaceuticalcomposition. Generally out of one hundred percent, this amount willrange from about 0.01% to 99% of the compound, preferably from about 5%to about 70%, most preferably from 10% to about 30%. A weight % is aratio by weight of active ingredient to total composition. Thus, forexample, 0.1 weight % is 0.1 grams of the compound per 100 grams oftotal composition.

The preparation of pharmaceutical compositions that contain an activecomponent is well understood in the art, for example, by mixing,granulating, or tablet-forming processes. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active agents are mixed with additives customary forthis purpose, such as vehicles, stabilizers, or inert diluents, andconverted by customary methods into suitable forms for administration,such as tablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic, or oily solutions and the like as detailed above.

For intravenous administration, glucuronic acid, L-lactic acid, aceticacid, citric acid or any pharmaceutically acceptable acid/conjugate basewith reasonable buffering capacity in the pH range acceptable forintravenous administration can be used as buffers. Sodium chloridesolution wherein the pH has been adjusted to the desired range witheither acid or base, for example, hydrochloric acid or sodium hydroxide,can also be employed. Typically, a pH range for the intravenousformulation can be in the range of from about 5 to about 12.

Subcutaneous formulations can be prepared according to procedures wellknown in the art at a pH in the range between about 5 and about 12,which include suitable buffers and isotonicity agents. They can beformulated to deliver a daily dose of the active agent in one or moredaily subcutaneous administrations. The choice of appropriate buffer andpH of a formulation, depending on solubility of one or more compounds tobe administered, is readily made by a person having ordinary skill inthe art. Sodium chloride solution wherein the pH has been adjusted tothe desired range with either acid or base, for example, hydrochloricacid or sodium hydroxide, can also be employed in the subcutaneousformulation. Typically, a pH range for the subcutaneous formulation canbe in the range of from about 5 to about 12.

Formulations of the invention suitable for oral administration can be inthe form of a solid, gel or liquid. For example, the formulation can bein the form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike. More specific examples of oral tablets include compressed,chewable lozenges and tablets that can be enteric-coated, sugarcoated orfilm-coated. Examples of capsules include hard or soft gelatin capsules.Granules and powders can be provided in non-effervescent or effervescentforms. Each can be combined with other ingredients known to thoseskilled in the art.

In certain embodiments, the compound described herein can be provided assolid dosage forms, preferably capsules or tablets. The tablets, pills,capsules, troches and the like can optionally contain one or more of thefollowing ingredients, or compounds of a similar nature: a binder; adiluent; a disintegrating agent; a lubricant; a glidant; a sweeteningagent; and a flavoring agent.

Examples of binders that can be used include, but are not limited to,microcrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, sucrose and starch paste.

Examples of lubricants that can be used include, but are not limited to,talc, starch, magnesium or calcium stearate, lycopodium and stearicacid.

Examples of diluents that can be used include, but are not limited to,lactose, sucrose, starch, kaolin, salt, mannitol and dicalciumphosphate.

Examples of glidants that can be used include, but are not limited to,colloidal silicon dioxide.

Examples of disintegrating agents that can be used include, but are notlimited to, crosscarmellose sodium, sodium starch glycolate, alginicacid, corn starch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose.

Examples of coloring agents that can be used include, but are notlimited to, any of the approved certified water soluble FD and C dyes,mixtures thereof; and water insoluble FD and C dyes suspended on aluminahydrate.

Examples of sweetening agents that can be used include, but are notlimited to, sucrose, lactose, mannitol and artificial sweetening agentssuch as sodium cyclamate and saccharin, and any number of spray-driedflavors.

Examples of flavoring agents that can be used include, but are notlimited to, natural flavors extracted from plants such as fruits andsynthetic blends of compounds that produce a pleasant sensation, suchas, but not limited to peppermint and methyl salicylate.

Examples of wetting agents that can be used include, but are not limitedto, propylene glycol monostearate, sorbitan monooleate, diethyleneglycol monolaurate and polyoxyethylene lauryl ether.

Examples of anti-emetic coatings that can be used include, but are notlimited to, fatty acids, fats, waxes, shellac, ammoniated shellac andcellulose acetate phthalates.

Examples of film coatings that can be used include, but are not limitedto, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethyleneglycol 4000 (PEG 4000) and cellulose acetate phthalate.

If oral administration is desired, the salt of the compound canoptionally be provided in a composition that protects it from the acidicenvironment of the stomach. For example, the composition can beformulated in an enteric coating that maintains its integrity in thestomach and releases the active compound in the intestine. Thecomposition can also be formulated in combination with an antacid orother such ingredient.

When the dosage unit form is a capsule, it can optionally additionallycomprise a liquid carrier such as a fatty oil. In addition, dosage unitforms can optionally additionally comprise various other materials thatmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents.

Compounds according to the present invention can also be administered asa component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup can optionally comprise, in addition to theactive compounds, sucrose as a sweetening agent and certainpreservatives, dyes and colorings and flavors.

The compounds described herein can also be mixed with other activematerials that do not impair the desired action, or with materials thatsupplement the desired action. For example, if a compound is used fortreating cancer, it can be used with other anti-cancer agents.

Examples of pharmaceutically acceptable carriers that can be included intablets comprising the compounds described herein include, but are notlimited to binders, lubricants, diluents, disintegrating agents,coloring agents, flavoring agents, and wetting agents. Enteric-coatedtablets, because of the enteric-coating, resist the action of stomachacid and dissolve or disintegrate in the neutral or alkaline intestines.Sugar-coated tablets can be compressed tablets to which different layersof pharmaceutically acceptable substances are applied. Film-coatedtablets can be compressed tablets that have been coated with polymers orother suitable coating. Multiple compressed tablets can be compressedtablets made by more than one compression cycle utilizing thepharmaceutically acceptable substances previously mentioned. Coloringagents can also be used in tablets. Flavoring and sweetening agents canbe used in tablets, and are especially useful in the formation ofchewable tablets and lozenges.

Examples of liquid oral dosage forms that can be used include, but arenot limited to, aqueous solutions, emulsions, suspensions, solutionsand/or suspensions reconstituted from non-effervescent granules andeffervescent preparations reconstituted from effervescent granules.

Examples of aqueous solutions that can be used include, but are notlimited to, elixirs and syrups. As used herein, elixirs refer to clear,sweetened, hydroalcoholic preparations. Examples of pharmaceuticallyacceptable carriers that can be used in elixirs include, but are notlimited to solvents. Particular examples of solvents that can be usedinclude glycerin, sorbitol, ethyl alcohol and syrup. As used herein,syrups refer to concentrated aqueous solutions of a sugar, for example,sucrose. Syrups can optionally further comprise a preservative.

Emulsions refer to two-phase systems in which one liquid is dispersed inthe form of small globules throughout another liquid. Emulsions canoptionally be oil-in-water or water-in oil emulsions. Examples ofpharmaceutically acceptable carriers that can be used in emulsionsinclude, but are not limited to non-aqueous liquids, emulsifying agentsand preservatives.

Examples of pharmaceutically acceptable substances that can be used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents.

Examples of pharmaceutically acceptable substances that can be used ineffervescent granules, to be reconstituted into a liquid oral dosageform, include organic acids and a source of carbon dioxide.

Coloring and flavoring agents can optionally be used in all of the abovedosage forms.

Particular examples of preservatives that can be used include glycerin,methyl and propylparaben, benzoic add, sodium benzoate and alcohol.

Particular examples of non-aqueous liquids that can be used in emulsionsinclude mineral oil and cottonseed oil.

Particular examples of emulsifying agents that can be used includegelatin, acacia, tragacanth, bentonite, and surfactants such aspolyoxyethylene sorbitan monooleate.

Particular examples of suspending agents that can be used include sodiumcarboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Diluentsinclude lactose and sucrose. Sweetening agents include sucrose, syrups,glycerin and artificial sweetening agents such as sodium cyclamate andsaccharin.

Particular examples of wetting agents that can be used include propyleneglycol monostearate, sorbitan monooleate, diethylene glycol monolaurateand polyoxyethylene lauryl ether.

Particular examples of organic acids that can be used include citric andtartaric acid.

Sources of carbon dioxide that can be used in effervescent compositionsinclude sodium bicarbonate and sodium carbonate. Coloring agents includeany of the approved certified water-soluble FD and C dyes, and mixturesthereof.

Particular examples of flavoring agents that can be used include naturalflavors extracted from plants such fruits, and synthetic blends ofcompounds that produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is preferablyencapsulated in a gelatin capsule. Such solutions, and the preparationand encapsulation thereof, are disclosed in U.S. Pat. Nos. 4,328,245;4,409,239; and 4,410,545, content of all of which is incorporated hereinby reference in their entirety. For a liquid dosage form, the solution,e.g., for example, in a polyethylene glycol, can be diluted with asufficient quantity of a pharmaceutically acceptable liquid carrier,e.g. water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations can be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g. propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. Nos. Re 28,819 and4,358,603, content of both of which is incorporated herein by referencein their entirety.

The present invention is also directed to compositions designed toadminister the compounds described herein by parenteral administration,generally characterized by injection, either subcutaneously,intramuscularly or intravenously. Injectables can be prepared in anyconventional form, for example as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions.

Examples of excipients that can be used in conjunction with injectablesaccording to the present invention include, but are not limited towater, saline, dextrose, glycerol or ethanol. The injectablecompositions can also optionally comprise minor amounts of non-toxicauxiliary substances 50 such as wetting or emulsifying agents, pHbuffering agents, stabilizers, solubility enhancers, and other suchagents, such as for example, sodium acetate, sorbitan monolaurate,triethanolamine oleate and cyclodextrins. Implantation of a slow releaseor sustained-release system, such that a constant level of dosage ismaintained is also contemplated herein. The percentage of activecompound contained in such parenteral compositions is highly dependenton the specific nature thereof, as well as the activity of the compoundand the needs of the subject.

Parenteral administration of the formulations includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as the lyophilized powders describedherein, ready to be combined with a solvent just prior to use, includinghypodermic tablets, sterile suspensions ready for injection, sterile dry

insoluble products ready to be combined with a vehicle just prior to useand sterile emulsions. The solutions can be either aqueous ornonaqueous.

When administered intravenously, examples of suitable carriers include,but are not limited to physiological saline or phosphate buffered saline(PBS), and solutions containing thickening and solubilizing agents, suchas glucose, polyethylene glycol, and polypropylene glycol and mixturesthereof.

Examples of pharmaceutically acceptable carriers that can optionally beused in parenteral preparations include, but are not limited to aqueousvehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents,buffers, antioxidants, local anesthetics, suspending and dispersingagents, emulsifying agents, sequestering or chelating agents and otherpharmaceutically acceptable substances.

Examples of aqueous vehicles that can optionally be used include SodiumChloride Injection, Ringers Injection, Isotonic Dextrose Injection,Sterile Water Injection, Dextrose and Lactated Ringers Injection.

Examples of nonaqueous parenteral vehicles that can optionally be usedinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil.

Antimicrobial agents in bacteriostatic or fungistatic concentrations canbe added to parenteral preparations, particularly when the preparationsare packaged in multiple-dose containers and thus designed to be storedand multiple aliquots to be removed. Examples of antimicrobial agentsthat can be used include phenols or cresols, mercurials, benzyl alcohol,chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters,thimerosal, benzalkonium chloride and benzethonium chloride.

Examples of isotonic agents that can be used include sodium chloride anddextrose. Examples of buffers that can be used include phosphate andcitrate. Examples of antioxidants that can be used include sodiumbisulfate. Examples of local anesthetics that can be used includeprocaine hydrochloride. Examples of suspending and dispersing agentsthat can be used include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Examples of emulsifying agentsthat can be used include Polysorbate 80 (TWEEN 80). A sequestering orchelating agent of metal ions include EDTA.

Pharmaceutical carriers can also optionally include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles andsodium hydroxide, hydrochloric acid, citric acid or lactic acid for pHadjustment.

The concentration of the compound in the parenteral formulation can beadjusted so that an injection administers a pharmaceutically effectiveamount sufficient to produce the desired pharmacological effect.

Injectables can be designed for local and systemic administration.

Unit-dose parenteral preparations can be packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration should be sterile, as is known and practiced in the art.

The compounds described herein can optionally be suspended in micronizedor other suitable form or can be derivatized to produce a more solubleactive product or to produce a prodrug. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease state and can be empiricallydetermined.

The compounds described herein can also be prepared as lyophilizedpowders, which can be reconstituted for administration as solutions,emulsions and other mixtures. The lyophilized powders can also beformulated as solids or gels.

Sterile, lyophilized powder can be prepared by dissolving the compoundin a sodium phosphate buffer solution containing dextrose or othersuitable excipient. Subsequent sterile filtration of the solutionfollowed by lyophilization under standard conditions known to those ofskill in the art provides the desired formulation. Briefly, thelyophilized powder can optionally be prepared by dissolving dextrose,sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose orother suitable agent, about 1-20%, preferably about 5 to 15%, in asuitable buffer, such as citrate, sodium or potassium phosphate or othersuch buffer known to those of skill in the art at, typically, aboutneutral pH. Then, a compound described herein is added to the resultingmixture, preferably above room temperature, more preferably at about30-35° C., and stirred until it dissolves. The resulting mixture isdiluted by adding more buffer to a desired concentration. The resultingmixture is sterile filtered or treated to remove particulates and toinsure sterility, and apportioned into vials for lyophilization. Eachvial can contain a single dosage or multiple dosages of the compounddescribed herein.

The compounds described herein can also be administered as topicalmixtures. Topical mixtures can be used for local and systemicadministration. The resulting mixture can be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The compounds described herein can be formulated as aerosols for topicalapplication, such as by inhalation (see, U.S. Pat. Nos. 4,044,126,4,414,209, and 4,364,923, which describe aerosols for delivery of asteroid useful for treatment inflammatory diseases, particularlyasthma). These formulations for administration to the respiratory tractcan be in the form of an aerosol or solution for a nebulizer, or as amicro fine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation will typically have diameters of less than 50 microns,preferably less than 10 microns.

The compounds described herein can also be formulated for local ortopical application, such as for topical application to the skin andmucous membranes, such as in the eye, in the form of gels, creams, andlotions and for application to the eye or for intracisternal orintraspinal application. Topical administration is contemplated fortransdermal delivery and also for administration to the eyes or mucosa,or for inhalation therapies. Nasal solutions of the compound alone or incombination with other pharmaceutically acceptable excipients can alsobe administered.

Depending upon the disease state being treated, other routes ofadministration, such as topical application, transdermal patches, andrectal administration, can also be used. For example, pharmaceuticaldosage forms for rectal administration are rectal suppositories,capsules and tablets for systemic effect. Rectal suppositories are usedherein mean solid bodies for insertion into the rectum that melt orsoften at body temperature releasing one or more pharmacologically ortherapeutically active ingredients. Pharmaceutically acceptablesubstances utilized in rectal suppositories are bases or vehicles andagents to raise the melting point. Examples of bases include cocoabutter (theobroma oil), glycerin-gelatin, carbowax, (polyoxyethyleneglycol) and appropriate mixtures of mono-, di- and triglycerides offatty acids. Combinations of the various bases can be used. Agents toraise the melting point of suppositories include spermaceti and wax.Rectal suppositories can be prepared either by the compressed method orby molding. The typical weight of a rectal suppository is about 2 to 3gm. Tablets and capsules for rectal administration can be manufacturedusing the same pharmaceutically acceptable substance and by the samemethods as for formulations for oral administration.

The invention is also directed to kits and other articles of manufacturefor treating infectious diseases or neurodegenerative diseases ordisorders. In one embodiment, a kit is provided that comprises acomposition comprising at least one compound disclosed herein incombination with instructions. The instructions can indicate the diseasestate for which the composition is to be administered, storageinformation, dosing information and/or instructions regarding how toadminister the composition. The kit can also comprise packagingmaterials. The packaging material can comprise a container for housingthe composition. The kit can also optionally comprise additionalcomponents, such as syringes for administration of the composition. Thekit can comprise the composition in single or multiple dose forms.

In another embodiment, an article of manufacture is provided thatcomprises a composition comprising at least one compound disclosedherein in combination with packaging materials. The packaging materialcan comprise a container for housing the composition. The container canoptionally comprise a label indicating the disease state for which thecomposition is to be administered, storage information, dosinginformation and/or instructions regarding how to administer thecomposition. The kit can also optionally comprise additional components,such as syringes for administration of the composition. The kit cancomprise the composition in single or multiple dose forms.

It is noted that the packaging material used in kits and articles ofmanufacture according to the present invention can form a plurality ofdivided containers such as a divided bottle or a divided foil packet.The container can be in any conventional shape or form as known in theart which is made of a pharmaceutically acceptable material, for examplea paper or cardboard box, a glass or plastic bottle or jar, are-sealable bag (for example, to hold a “refill” of tablets forplacement into a different container), or a blister pack with individualdoses for pressing out of the pack according to a therapeutic schedule.The container that is employed will depend on the exact dosage forminvolved, for example a conventional cardboard box would not generallybe used to hold a liquid suspension. It is feasible that more than onecontainer can be used together in a single package to market a singledosage form. For example, tablets can be contained in a bottle that isin turn contained within a box. Typically the kit includes directionsfor the administration of the separate components. The kit form isparticularly advantageous when the separate components are preferablyadministered in different dosage forms (e.g., oral, topical, transdermaland parenteral), are administered at different dosage intervals, or whentitration of the individual components of the combination is desired bythe prescribing physician.

One particular example of a kit according to the present invention is aso-called blister pack. Blister packs are well known in the packagingindustry and are being widely used for the packaging of pharmaceuticalunit dosage forms (tablets, capsules, and the like). Blister packsgenerally consist of a sheet of relatively stiff material covered with afoil of a preferably transparent plastic material. During the packagingprocess recesses are formed in the plastic foil. The recesses have thesize and shape of individual tablets or capsules to be packed or canhave the size and shape to accommodate multiple tablets and/or capsulesto be packed. Next, the tablets or capsules are placed in the recessesaccordingly and the sheet of relatively stiff material is sealed againstthe plastic foil at the face of the foil which is opposite from thedirection in which the recesses were formed. As a result, the tablets orcapsules are individually sealed or collectively sealed, as desired, inthe recesses between the plastic foil and the sheet. Preferably thestrength of the sheet is such that the tablets or capsules can beremoved from the blister pack by manually applying pressure on therecesses whereby an opening is formed in the sheet at the place of therecess. The tablet or capsule can then be removed via said opening.

Another specific embodiment of a kit is a dispenser designed to dispensethe daily doses one at a time in the order of their intended use.Preferably, the dispenser is equipped with a memory-aid, so as tofurther facilitate compliance with the regimen. An example of such amemory-aid is a mechanical counter that indicates the number of dailydoses that has been dispensed. Another example of such a memory-aid is abattery-powered micro-chip memory coupled with a liquid crystal readout,or audible reminder signal which, for example, reads out the date thatthe last daily dose has been taken and/or reminds one when the next doseis to be taken.

The compounds described herein can be used or administrated to a subjectin combination with another compound, e.g., a pharmaceutically activeagent or treatment modality for a particular indication. Exemplarypharmaceutically active compound include, but are not limited to, thosefound in Harrison's Principles of Internal Medicine, 13^(th) Edition,Eds. T. R. Harrison et al. McGraw-Hill N.Y., N.Y.; Physicians' DeskReference, 50^(th) Edition, 1997, Oradell N.J., Medical Economics Co.;Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990; current edition of Goodman and Oilman's ThePharmacological Basis of Therapeutics; and current edition of The MerckIndex, the complete contents of all of which are incorporated herein byreference.

Wide variety compounds, e.g., therapeutic agents can have an additive orsynergistic effect with the compounds described herein. Such compoundscan additively or synergistically combine with the compounds describedherein in the methods disclosed herein, e.g., to differentiate stemcells and/or treat a neurodegenerative disease or disorder in a subject.

The inventors have discovered that the combination of compoundsdisclosed herein, e.g., 7-chloro-4-aminoquinoline compounds withforskoline unexpectedly, surprisingly shows a synergistic effect onstimulating the transcriptional activity through the ligand bindingdomain of Nurr1 and enhancing the contrasting dual function of Nurr1.

The term “synergistic” as used herein is defined to mean a combinationof components wherein the activity of the combination is greater thanthe additive of the individual activities of each component of thecombination. In some embodiments, the activity of the combination is atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 1-fold, at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least50-fold, at least 100-fold or greater than the additive of theindividual activities of each component of the combination.

In some embodiments, a compound disclosed herein can be used incombination with a dopamine agonist. Without wishing to be bound by atheory, it is believed that the combination of a compound disclosedherein with a dopamine agonist shows a synergistic effect on stimulatingthe transcriptional activity through the ligand binding domain of Nurr1and enhancing the contrasting dual function of Nurr1.

As used herein, the term “dopamine agonist” refers to compounds thatactivate and/or stimulate one or more dopamine receptors and/or increaselevels of dopamine (such as L-dopa or drugs which inhibit dopaminemetabolism) and/or stimulate a dopamine signaling pathway and/or reducelevels of norepinephrine, and/or inhibit a norepinephrine signalingpathway. The term “dopamine agonist” also includes analogs of dopaminemolecules which exhibit at least some biological activity in common withnative human dopamine receptors. As such, the term “dopamine agonist”encompasses dopaminergic agents. As used herein the term “dopaminergicagent” refers to compounds which mimic the action of dopamine.Accordingly, the term dopaminergic agent is intended to encompassdopamine, derivatives of dopamine, and compounds which have dopaminelike actions on dopamine receptors. Exemplary analogs of dopamineinclude the ergolines and the aporphines such apomorphine, pergolide,bromocriptine and lisuride). Dopamine agonists are primarily used forthe treatment of Parkinson's disease due to their neuroprotectiveeffects on dopaminergic neurons.

Without wishing to be bound by a theory, a dopamine agonist can act viaone of several pathways. For example, a dopamine agonist can activate orpotentiate D1 dopamine receptors and/or Dj-like receptors such as D1 andD5 dopamine receptors and/or D2 dopamine receptors (e.g., D2, D2 shortand D2 long receptors, D4, and D4 dopamine receptors) and/or D3 dopaminereceptors and/or D4 dopamine receptors. A dopamine agonist can act byinhibiting one or more enzyme involved in biosynthesis and/ortransformation and/or breakdown of dopamine.

Exemplary dopamine agonists include, but are not limited to,L-3,4-dihydroxyphenylalanine (L-Dopa);(−)-7-{[2-(4-Phenylpiperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol;(+)-4-propyl-9-hydroxynaphthoxazine ((+)PHNO);(E)-1-aryl-3-(4-pyridinepiperazin-1-yl)propanone oximes;(R)-3-(4-Propylmorpholin-2-yl)phenol (PF-219,061); (R,R)—S32504;2-(N-phenylethyl-N-propylamino)-5-hydroxytetralin;2-bromo-a-ergocriptine (bromocriptine);5,6,7,8-Tetrahydro-6-(2-propen-1-yl)-4H-thiazolo[4,5-d]azepin-2-amine(BHT-920); 5-HT uptake inhibitor; 5-HT-1A agonists (such as roxindole);6-Br-APB; 6-methyl-8-a-(N-acyl)amino-9-ergoline;6-methyl-8-a-(N-phenyl-acety)amino-9-ergoline;6-methyl-8β-carbobenzyloxy-aminoethyl-10-a-ergoline;7,8-Dihydroxy-5-phenyl-octahydrobenzo[h]isoquinoline;8-acylaminoergoline; 9,10-dihydroergocomine; a2-adrenergic antagonist(such as terguride); A-412,997; A-68,930; A-77,636; A-86,929; ABT-670;ABT-724; AF-14; alaptide; amisulpride; anyD-2-halo-6-alkyl-8-substituted ergoline; Aplindore; Apomorphine;Aripiprazole (Abilify in USA); benzazepine analogs; BP-897;Bromocriptine; bromocriptine mesylate; Cabergoline;cis-8-Hydroxy-3-(n-propyl)-1,2,3a,4,5,9b-hexahydro-1H- andtrans-N-{4-[4-(2,3-Dichlorophenyl)-1-piperazinyl]cyclohexyl}-3-methoxybenzamide;clozapine; COMT inhibitors (such as CGP-28014, entacapone andtolcapone); CP-226,269; CP-96,345; CY-208,243;D-2-bromo-6-methyl-8-cyanomethylergoline; Dihydrexidine;dihydro-alpha-ergocriptine; dihydro-alpha-ergotoxine;dihydroergocriptine; dihydroergocryptine; dihydroergotoxine (hydergine);Dinapsoline; Dinoxyline; domperidone; Dopamine; dopamine D1 receptoragonists; dopamine D2 receptor agonists; dopamine D3 receptor agonists;dopamine D4 receptor agonists; dopamine D5 receptor agonists; dopamineuptake inhibitors (such as GBR-12909, GBR-13069, GYKI-52895, andNS-2141); doprexin; Doxanthrine; ER-230; erfotoxine; Ergocornine;ergoline derivatives; ergot alkaloid derivatives; eticlopride;etisulergine; FAUC 299; FAUC 316; Fenoldopam; Flibanserin; haloperidol;iloperidone; levodopa; Lisuride; lisuride; LSD; LU111995; mazapertine;Methylphenidate; monoamine oxidase-B inhibitors (such as selegiline,N-(2butyl)-N-methylpropargylamine, N-methyl-N-(2-pentyl) propargylamine,AGN-1133, ergot derivatives, lazabemide, LU-53439, MD-280040 andmofegiline); N-0434; Naxagolide; olanzapine; opiate receptor agonists(such as NIH-10494); PD-118,440; PD-168,077; Pergolide (such as A-68939,A-77636, dihydrexine, and SKF-38393); PIP3EA; piribedil; Piribedil;Pramipexole; Quinagolide; Quinelorane; Quinpirole; racemictrans-10,11-dihydroxy 5,6,6a, 7,8,12b-hexahydro and related benzazepineanalogs; raclopride; remoxipride; risperidone; Ro10-5824; Ropinirole;Rotigotine; Salvinorin A; SDZ-HDC-912; sertindole; SKF-38,393;SKF-75,670; SKF-81,297; SKF-82,526 (fenoldopam); SKF-82,598; SKF-82,957;SKF-82,958; SKF-38,393; SKF-77,434; SKF-81,297; SKF-82,958; SKF-89,145;SKF-89,626; spiperone; spiroperidol; sulpride; sumanirole; Talipexole;Terguride; tropapride; WAY-100635; YM 09151-2; zetidoline; β-adrenergicreceptor agonists; cabergoline; bromocriptine; pergolide; talipexole;ropinirole; pramipexole; and analogs, derivatives, enantiomers,metabolites, prodrugs, and pharmaceutically acceptable salts thereof.

Exemplary beta-3 adrenergic receptor agonists include, but are notlimited to, DPDMS; dopexamine; AJ-9677; AZ-40140; BMS187413; BMS-194449;BMS-210285; BRL-26830A; BRL-28410; BRL-35135; BRL-37344; CGP 12177;CL-316243; CP-114271; CP-331648; CP-331679; D-7114; FR-149175; GW-2696;GW-427353; ICI-198157; L-750355; L-796568; LY-377604; N-5984; SB-226552;SR-58611A; SR-59062A; SWR0342SA; ZD-2079; and analogs, derivatives,enantiomers, metabolites, prodrugs, and pharmaceutically acceptablesalts thereof.

In some embodiments, the dopamine agonist inhibits the dopaminebeta-hydroxylase. Dopamine beta-hydroxylase converts dopamine tonorepinephrine. Thus, by inhibiting dopamine beta-hydroxylase,intracellular dopamine is increased while norepinephrine is decreased.

Exemplary inhibitors of DBH include, but are not limited to fusaricacid; 1,1′,1″,1′″-[disulfanediylbis-(carbonothioylnitrilo)]tetraethane(disulflram); 2-Hydroxy-2,4,6-cycloheptatrien-1-one (tropolone, alsoreferred to as 2-Hydroxytropone or Purpurocatechol);5-(aminomethyl)-1-[(2S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]-1,3-dihydro-2H-imidazole-2-thione(Nepicastat, INN, or SYN117)); 1-(4-hydroxybenzyl)imidazole-2-thiol;FLA-63; diethyidithiocarbamate; betachlorophenethylamine;4-hydroxybenzyl cyanide; 2-halo-3(p-hydroxyphenyl)-1-propene;1-phenyl-1-propyne; 2-phenylallylamine; 2-(2-thienyl)allylamine;2-thiophene-2(2-thienyl)allylamine; 3-phenylpropargylamine; 1-phenyl-1(aminoethyl)ethane; N-(trifluoroacetyl)phenyl(aminoethyl) ethane;5-picolinic acid substituted with an alkyl group containing up to 6carbon atoms; 5-picolinic acid substituted with a halo alkyl groupcontaining up to 6 carbon atoms; and analogs, derivatives, enantiomers,metabolites, prodrugs, and pharmaceutically acceptable salts thereof.Other inhibitors of dopamine beta-hydroxylase include, but are notlimited to U.S. Pat. Nos. 4,487,761; 4,634,711; 4,719,223; 4,743,613;4,749,717; 4,761,415; 4,762,850; 4,798,843; 4,810,800; 4,835,154;4,839,371; 4,859,779; 4,876,266; 4,882,348; 4,906,668; 4,935,438;4,963,568; 4,992,459; 5,100,912; 5,189,052; 5,597,832; 6,407,137;6,559,186; 7,125,904; 7,576,081, content of all of which is hereinincorporated by reference in their entirety.

Cabergoline (Dostinex®) is a long-acting ergot derivative agonist with ahigh affinity for D2 receptors. Bromocriptine (Parlodel®) is an ergotalkaloid dopamine receptor agonist. It is a strong D2 receptor agonistand a weak DI receptor antagonist. It stimulates both pre- andpost-synaptic receptors. Pergolide (Permax®) is a semisynthetic, clavineergot derivative dopamine agonist. In contrast to bromocriptine, it is astrong D2 receptor agonist and a weak D1 receptor agonist. Ropinirole(Requip®) is a potent, non-ergoline dopamine agonist. Pramipexole(Mirapex®) is a synthetic amino-benzothiazol derivative and a non-ergotD2/D3 agonist. Quinagolide (1-propylbenzo[g]quinolin-3-yl)-Norprolac®;formerly CV 205-502) is another, non-ergot, non-ergoline, benzoquinolinedopaminergic agonist that blocks prolactin release.

For in vivo methods, a compound disclosed herein can be co-adminsteredwith the dopamine agonist. For example, for treating neurodegenerativedisorders, the compound disclosed herein can be co-adminstered with thedopamine agonist.

For treating inflammation or inflammation associated disorders, acompound disclosed herein can be co-adminstered with an agent known inthe art for treatment of inflammation or inflammation associateddisorders or infections. Exemplary anti-inflammatory agents include, butare not limited to, non-steroidal anti-inflammatory drugs (NSAIDs—suchas aspirin, ibuprofen, or naproxen, coricosteroids (such as prednisone),anti-malarial medication (such as hydrochloroquine), methotrexrate,sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamise,mycophenolate, dexamethasone, rosiglitazone, prednisolone,corticosterone, budesonide, estrogen, estrodiol, sulfasalazine,fenfibrate, provastatin, simvastatin, proglitazone, acetylsalicylicacid, mycophenolic acid, mesalamine, and analogs, derivatives, prodrugs,and pharmaceutically acceptable salts thereof.

The term “co-administering,” “co-administration,” or “co-administer”refers to the administration of a compound disclosed herein and a secondcompound, e.g. a dopamine agonist, forskolin, or colfosin, wherein thecompound disclosed herein and the second compound can be administeredsimultaneously, or at different times, as long as they work additivelyor synergistically to increase stem cell differentiation.

Without limitations, the compound disclosed herein and the secondcompound can be administered in the same formulation or in separateformulations. When administered in separate formulations, the compounddisclosed herein and the second compound can be administered within anytime of each other. For example, the compounds can be administeredwithin 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours,1 hours, 45 minutes, 30 minute, 25 minutes, 20 minutes, 15 minutes, 10minutes, 5 minutes or less of each other. When administered in separateformulations, either compound can be administered first.

Additionally, co-administration does not require the two compounds to beadministered by the same route. As such, each can be administeredindependently or as a common dosage form. Further, the two compounds canbe administered in any ratio to each other by weight or moles. Forexample, two compounds can be administered in a ratio of from about50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1.75, 1.5:1,or 1.25:1 to 1:1.25, 1:1.5, 1.75, 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20,1:20, 1:30, 1:40, or 1:50. The ratio can be based on the effectiveamount of either compound.

In some embodiments, a compound disclosed herein can be co-adminsteredwith forskolin or colfosin.

In some embodiments, amodiaquine or chloroquine can be co-adminsteredwith forskolin or colfosin. In some other embodiments,

In some embodiments, a compound disclosed herein is co-adminsteredamodiaquine or chloroquine can be co-adminstered with a dopamineagonist.

Without limitation, methods comprising co-administering a compounddisclosed herein can provide novel mechanism-based neuroprotectivetherapy that could have disease-modifying effects in (neuro)inflammatorydiseases such as PD and AD as well as auto-immune diseases such asrheumatoid arthritis and lupus disease. Furthermore, since the currentpharmacological treatments of PD by dopamine agonists such as L-Dopaleads to severe side effects while AQ or CQ did not induce any such sideeffects, their combined use will reduce or eliminate the side effects bydopamine agonists.

The invention can be defined by any of the following numberedparagraphs:

-   1. A compound of formula (I):

-   -   wherein:    -   X, Y, and Z are independently CR¹⁸, N, O, or S;    -   one of Z¹ and Z² is N and the other is CR¹⁴;    -   R and R¹⁰-R¹⁸ independently H, linear or branched alkyl, linear        or branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted; and    -   L is a linker.

-   2. A compound of formula (II):

-   -   wherein:    -   X and Y are independently CR¹⁸, N, O, or S;    -   A and B are independently CR¹⁸, N, O, or S;    -   one of Z¹ and Z² is N and the other is CR¹⁴;    -   R and R¹⁰-R¹⁸ independently H, linear or branched alkyl, linear        or branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted;    -   ----- forms a cyclyl, heterocyclyl, aryl, or heteroaryl, each of        which can be optionally substituted; and    -   L is a linker.

-   3. A compound of formula (III):

-   -   wherein:    -   A and B are independently CR¹⁸ or N;    -   one of Z¹ and Z² is N and the other is CR¹⁴;    -   R and R¹⁰-R¹⁸ independently H, linear or branched alkyl, linear        or branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted; and    -   L is a linker.

-   4. A compound of formula (IV):

-   -   wherein:    -   X and Z are independently CR¹⁸, N, O, or S;    -   one of Z¹ and Z² is N and the other is CR¹⁴;    -   R and R¹¹-R¹⁸ independently H, linear or branched alkyl, linear        or branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted;    -   ----- forms a cyclyl, heterocyclyl, aryl, or heteroaryl, each of        which can be optionally substituted; and    -   L is a linker.

-   5. A compound of formula (V):

-   -   wherein:    -   X, Y, and Z are independently CR¹⁸, N, O, or S;    -   one of Z¹ and Z² is N and the other is CR¹⁴;    -   R and R¹⁰-R¹⁸ independently H, linear or branched alkyl, linear        or branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted; and    -   ----- forms a cyclyl, heterocyclyl, aryl, or heteroaryl, each of        which can be optionally substituted.

-   6. A compound of formula (VI):

-   -   wherein:    -   X, Y, and Z are independently CR¹⁸, N, O, or S;    -   R²¹-R²⁸ independently H, linear or branched alkyl, linear or        branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted;    -   ----- forms a cyclyl, heterocyclyl, aryl, or heteroaryl, each of        which can be optionally substituted; and    -   L is a linker.

-   7. A compound of formula (VII):

-   -   wherein:    -   X, Y, and Z are independently CR¹⁸, N, O, or S;    -   one of Z¹ and Z² is N and the other is CR¹⁴;    -   R and R¹⁰-R²² independently H, linear or branched alkyl, linear        or branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted.

-   8. A compound of formula (VIII):

-   -   wherein:    -   X, Y, and Z are independently CR¹⁸, N, O, or S;    -   one of Z¹ and Z² is N and the other is CR¹⁴;    -   R and R¹⁰-R²¹ independently H, linear or branched alkyl, linear        or branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted.

-   9. A compound of formula (IX):

-   -   wherein:    -   X, Y, and Z are independently CR¹⁸, N, O, or S;    -   one of Z¹ and Z² is N and the other is CR¹⁴;    -   R and R¹⁰-R²² independently H, linear or branched alkyl, linear        or branched alkenyl, linear or branched alkynyl, cyclyl,        heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,        alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino,        alkylamino, thiol, or alkylthio, each of which can be optionally        substituted.

-   10. A compound selected from the group consisting of

-   -   and any combinations thereof.

-   11. A pharmaceutical composition comprising a compound of any of    paragraphs 1-10 and a pharmaceutically acceptable carrier.

-   12. A composition comprising a compound of any of paragraphs 1-10    and stem cells.

-   13. The composition of paragraph 12, wherein the stem cells are    human embryonic stem cells.

-   14. A method of treating a disease state when a decreased Nurr1    activity contributes to the pathology or symptomology of the    disease, the method comprising administering a therapeutically    effective amount of a compound of any of paragraphs 1-10 to a    subject in need thereof.

-   15. A method of treating a disease state when a Nurr1 hypoactivity    contributes to the pathology or symptomology of the disease, the    method comprising administering a therapeutically effective amount    of a compound of any of paragraphs 1-10 to a subject in need    thereof.

-   16. A method of treating inflammation or inflammation associated    disease or disorder in a subject, the method comprising    administering a therapeutically effective amount of a compound of    any of paragraphs 1-10 to a subject in need thereof.

-   17. A method of treating a neurodegenerative disease or disorder in    a subject, the method comprising administering a therapeutically    effective amount of a compound of any of paragraphs 1-10 to a    subject in need thereof.

-   18. The method of paragraph 17, wherein the neurodegenerative    disease or disorder is Parkinson's disease.

-   19. The method of paragraph 17 or 18, further comprising    co-administering forskolin, colforsin, or a dopamine agonist to the    subject.

-   20. The method of paragraph 19, wherein the dopamine agonist is    L-DOPA.

-   21. The method of any of paragraphs 14-20, wherein the    therapeutically effective amount is an amount sufficient to activate    Nurr1 in cells of the subject.

-   22. A method of inhibiting a neurodegenerative disease or disorder    in a subject, the method comprising co-administering: (i) a    composition comprising stem cells; and (ii) a compound of any of    paragraphs 1-10 in an amount sufficient to induce differentiation of    the stem cells.

-   23. The method of paragraph 22, wherein the stem cells are human    embryonic stem cells.

-   24. A method for inducing differentiation of a stem cell into a    dopaminergic neuron, the method comprising contacting said cell with    a compound of any of paragraphs 1-10 in an amount sufficient to    cause differentiation of said stem cell.

-   25. The method of paragraph 24, wherein the stem cell is a human    embryonic stem cell.

-   26. A kit comprising a compound of any of paragraphs 1-10 and    instructions for administering said compound to a subject diagnosed    with or at risk of developing a neurodegenerative disease or    disorder.

-   27. The kit of paragraph 26, further comprising a stem cell.

-   28. A method of treating a disease state for which a dopamine    agonist is a useful treatment, the method comprising administering a    therapeutically effective amount of a compound of any of paragraphs    1-10 to a subject in need thereof

-   29. The method of paragraph 28, wherein the disease state is    restless leg syndrome.

Some Selected Definitions

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means at least two standarddeviations (2SD) away from a reference level. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true.

As used herein, the term “aliphatic” means a moiety characterized by astraight or branched chain arrangement of constituent carbon atoms andcan be saturated or partially unsaturated with one or more (e.g., one,two, three, four, five or more) double or triple bonds.

As used herein, the term “alicyclic” means a moiety comprising anonaromatic ring structure. Alicyclic moieties can be saturated orpartially unsaturated with one or more double or triple bonds. Alicyclicmoieties can also optionally comprise heteroatoms such as nitrogen,oxygen and sulfur. The nitrogen atoms can be optionally quaternized oroxidized and the sulfur atoms can be optionally oxidized. Examples ofalicyclic moieties include, but are not limited to moieties with C₃-C₈rings such as cyclopropyl, cyclohexane, cyclopentane, cyclopentene,cyclopentadiene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane,cycloheptene, cycloheptadiene, cyclooctane, cyclooctene, andcyclooctadiene.

As used herein, the term, “aromatic” means a moiety wherein theconstituent atoms make up an unsaturated ring system, all atoms in thering system are sp² hybridized and the total number of pi electrons isequal to 4n+2. An aromatic ring can be such that the ring atoms are onlycarbon atoms (e.g., aryl) or can include carbon and non-carbon atoms(e.g., heteroaryl).

As used herein, the term “alkyl” means a straight or branched, saturatedaliphatic radical having a chain of carbon atoms. C_(x) alkyl andC_(x)-C_(y)alkyl are typically used where X and Y indicate the number ofcarbon atoms in the chain. For example, C₁-C₆alkyl includes alkyls thathave a chain of between 1 and 6 carbons (e.g., methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, neopentyl,hexyl, and the like). Alkyl represented along with another radical(e.g., as in arylalkyl) means a straight or branched, saturated alkyldivalent radical having the number of atoms indicated or when no atomsare indicated means a bond, e.g., (C₆-C₁₀)aryl(C₀-C₃)alkyl includesphenyl, benzyl, phenethyl, 1-phenylethyl 3-phenylpropyl, and the like.Backbone of the alkyl can be optionally inserted with one or moreheteroatoms, such as N, O, or S.

In preferred embodiments, a straight chain or branched chain alkyl has30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straightchains, C3-C30 for branched chains), and more preferably 20 or fewer.Likewise, preferred cycloalkyls have from 3-10 carbon atoms in theirring structure, and more preferably have 5, 6 or 7 carbons in the ringstructure. The term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having one or more substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

Substituents of a substituted alkyl may include halogen, hydroxy, nitro,thiols, amino, azido, imino, amido, phosphoryl (including phosphonateand phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyland sulfonate), and silyl groups, as well as ethers, alkylthios,carbonyls (including ketones, aldehydes, carboxylates, and esters),—CF3, —CN and the like.

As used herein, the term “alkenyl” refers to unsaturated straight-chain,branched-chain or cyclic hydrocarbon radicals having at least onecarbon-carbon double bond. C_(x) alkenyl and C_(x)-C_(y)alkenyl aretypically used where X and Y indicate the number of carbon atoms in thechain. For example, C₂-C₆alkenyl includes alkenyls that have a chain ofbetween 1 and 6 carbons and at least one double bond, e.g., vinyl,allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl,2-methylallyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, and the like). Alkenylrepresented along with another radical (e.g., as in arylalkenyl) means astraight or branched, alkenyl divalent radical having the number ofatoms indicated. Backbone of the alkenyl can be optionally inserted withone or more heteroatoms, such as N, O, or S.

As used herein, the term “alkynyl” refers to unsaturated hydrocarbonradicals having at least one carbon-carbon triple bond. C_(x) alkynyland C_(x)-C_(y)alkynyl are typically used where X and Y indicate thenumber of carbon atoms in the chain. For example, C₂-C₆alkynyl includesalkynyls that have a chain of between 1 and 6 carbons and at least onetriple bond, e.g., ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,isopentenyl, 1,3-hexa-diyn-yl, n-hexynyl, 3-pentynyl, 1-hexen-3-ynyl andthe like. Alkynyl represented along with another radical (e.g., as inarylalkynyl) means a straight or branched, alkynyl divalent radicalhaving the number of atoms indicated. Backbone of the alkynyl can beoptionally inserted with one or more heteroatoms, such as N, O, or S.

The terms “alkylene,” “alkenylene,” and “alkynylene” refer to divalentalkyl, alkelyne, and alkynylene” radicals. Prefixes C_(x) andC_(x)-C_(y) are typically used where X and Y indicate the number ofcarbon atoms in the chain. For example, C₁-C₆alkylene includesmethylene, (—CH₂—), ethylene (—CH₂CH₂—), trimethylene (—CH₂CH₂CH₂—),tetramethylene (—CH₂CH₂CH₂CH₂—), 2-methyltetramethylene(—CH₂CH(CH₃)CH₂CH₂—), pentamethylene (—CH₂CH₂CH₂CH₂CH₂—) and the like).

As used herein, the term “alkylidene” means a straight or branchedunsaturated, aliphatic, divalent radical having a general formula═CR_(a)R_(b). C_(x) alkylidene and C_(x)-C_(y)alkylidene are typicallyused where X and Y indicate the number of carbon atoms in the chain. Forexample, C₂-C₆alkylidene includes methylidene (═CH₂), ethylidene(═CHCH₃), isopropylidene (═C(CH₃)₂), propylidene (═CHCH₂CH₃), allylidene(═CH—CH═CH₂), and the like).

The term “heteroalkyl”, as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom. Suitable heteroatoms include, butare not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorousand sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized. Heteroalkyls can be substituted as defined abovefor alkyl groups.

As used herein, the term “halogen” or “halo” refers to an atom selectedfrom fluorine, chlorine, bromine and iodine. The term “halogenradioisotope” or “halo isotope” refers to a radionuclide of an atomselected from fluorine, chlorine, bromine and iodine.

A “halogen-substituted moiety” or “halo-substituted moiety”, as anisolated group or part of a larger group, means an aliphatic, alicyclic,or aromatic moiety, as described herein, substituted by one or more“halo” atoms, as such terms are defined in this application. Forexample, halo-substituted alkyl includes haloalkyl, dihaloalkyl,trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted(C₁-C₃)alkyl includes chloromethyl, dichloromethyl, difluoromethyl,trifluoromethyl, 2,2,2-trifluoroethyl, perfluoroethyl,2,2,2-trifluoro-1,1-dichloroethyl, and the like).

The term “aryl” refers to monocyclic, bicyclic, or tricyclic fusedaromatic ring system. C_(x) aryl and C_(x)-C_(y)aryl are typically usedwhere X and Y indicate the number of carbon atoms in the ring system.Exemplary aryl groups include, but are not limited to, benzyl, phenyl,naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl,phenyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl,benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl,carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,2H,6H-1,5,2-dithiazinyl dihydrofuro[2,3 b]tetrahydrofuran, furanyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl,phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl and xanthenyl, and the like. In someembodiments, 1, 2, 3, or 4 hydrogen atoms of each ring can besubstituted by a substituent.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively. C_(x)heteroaryl and C_(x)-C_(y)heteroaryl are typically used where X and Yindicate the number of carbon atoms in the ring system. Heteroarylsinclude, but are not limited to, those derived from benzo[b]furan,benzo[b] thiophene, benzimidazole, imidazo[4,5-c]pyridine, quinazoline,thieno[2,3-c]pyridine, thieno[3,2-b]pyridine, thieno[2, 3-b]pyridine,indolizine, imidazo[1,2a]pyridine, quinoline, isoquinoline, phthalazine,quinoxaline, naphthyridine, quinolizine, indole, isoindole, indazole,indoline, benzoxazole, benzopyrazole, benzothiazole,imidazo[1,5-a]pyridine, pyrazolo[1,5-a]pyridine,imidazo[1,2-a]pyrimidine, imidazo[1,2-c]pyrimidine,imidazo[1,5-a]pyrimidine, imidazo[1,5-c]pyrimidine,pyrrolo[2,3-b]pyridine, pyrrolo[2,3c]pyridine, pyrrolo[3,2-c]pyridine,pyrrolo[3,2-b]pyridine, pyrrolo[2,3-d]pyrimidine,pyrrolo[3,2-d]pyrimidine, pyrrolo [2,3-b]pyrazine,pyrazolo[1,5-a]pyridine, pyrrolo[1,2-b]pyridazine,pyrrolo[1,2-c]pyrimidine, pyrrolo[1,2-a]pyrimidine,pyrrolo[1,2-a]pyrazine, triazo[1,5-a]pyridine, pteridine, purine,carbazole, acridine, phenazine, phenothiazene, phenoxazine,1,2-dihydropyrrolo[3,2,1-hi]indole, indolizine, pyrido[1,2-a]indole,2(1H)-pyridinone, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Someexemplary heteroaryl groups include, but are not limited to, pyridyl,furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl orthienyl, pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl,naphthyridinyl, 2-amino-4-oxo-3,4-dihydropteridin-6-yl,tetrahydroisoquinolinyl, and the like. In some embodiments, 1, 2, 3, or4 hydrogen atoms of each ring may be substituted by a substituent.

The term “cyclyl” or “cycloalkyl” refers to saturated and partiallyunsaturated cyclic hydrocarbon groups having 3 to 12 carbons, forexample, 3 to 8 carbons, and, for example, 3 to 6 carbons. C_(x)cyclyland C_(x)-C_(y)cylcyl are typically used where X and Y indicate thenumber of carbon atoms in the ring system. The cycloalkyl groupadditionally can be optionally substituted, e.g., with 1, 2, 3, or 4substituents. C₃-C₁₀cyclyl includes cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, 2,5-cyclohexadienyl, cycloheptyl,cyclooctyl, bicyclo[2.2.2]octyl, adamantan-1-yl, decahydronaphthyl,oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, 2-oxobicyclo[2.2.1]hept-1-yl, and the like.

Aryl and heteroaryls can be optionally substituted with one or moresubstituents at one or more positions, for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or thelike.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively). C_(x)heterocyclyl andC_(x)-C_(y)heterocyclyl are typically used where X and Y indicate thenumber of carbon atoms in the ring system. In some embodiments, 1, 2 or3 hydrogen atoms of each ring can be substituted by a substituent.Exemplary heterocyclyl groups include, but are not limited topiperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl,piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl,perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl,1,4-dioxanyl and the like.

The terms “bicyclic” and “tricyclic” refers to fused, bridged, or joinedby a single bond polycyclic ring assemblies.

The term “cyclylalkylene” means a divalent aryl, heteroaryl, cyclyl, orheterocyclyl.

As used herein, the term “fused ring” refers to a ring that is bonded toanother ring to form a compound having a bicyclic structure when thering atoms that are common to both rings are directly bound to eachother. Non-exclusive examples of common fused rings include decalin,naphthalene, anthracene, phenanthrene, indole, furan, benzofuran,quinoline, and the like. Compounds having fused ring systems can besaturated, partially saturated, cyclyl, heterocyclyl, aromatics,heteroaromatics, and the like.

As used herein, the term “carbonyl” means the radical —C(O)—. It isnoted that the carbonyl radical can be further substituted with avariety of substituents to form different carbonyl groups includingacids, acid halides, amides, esters, ketones, and the like.

The term “carboxy” means the radical —C(O)O—. It is noted that compoundsdescribed herein containing carboxy moieties can include protectedderivatives thereof, i.e., where the oxygen is substituted with aprotecting group. Suitable protecting groups for carboxy moietiesinclude benzyl, tert-butyl, and the like.

The term “cyano” means the radical —CN.

The term, “heteroatom” refers to an atom that is not a carbon atom.Particular examples of heteroatoms include, but are not limited tonitrogen, oxygen, sulfur and halogens. A “heteroatom moiety” includes amoiety where the atom by which the moiety is attached is not a carbon.Examples of heteroatom moieties include —N═, —NR^(N)—, —N⁺(O⁻)═, —O—,—S— or —S(O)₂—, —OS(O)₂—, and —SS—, wherein R^(N) is H or a furthersubstituent.

The term “hydroxy” means the radical —OH.

The term “imine derivative” means a derivative comprising the moiety—C(NR)—, wherein R comprises a hydrogen or carbon atom alpha to thenitrogen.

The term “nitro” means the radical —NO₂.

An “oxaaliphatic,” “oxaalicyclic”, or “oxaaromatic” mean an aliphatic,alicyclic, or aromatic, as defined herein, except where one or moreoxygen atoms (—O—) are positioned between carbon atoms of the aliphatic,alicyclic, or aromatic respectively.

An “oxoaliphatic,” “oxoalicyclic”, or “oxoaromatic” means an aliphatic,alicyclic, or aromatic, as defined herein, substituted with a carbonylgroup. The carbonyl group can be an aldehyde, ketone, ester, amide,acid, or acid halide.

As used herein, the term, “aromatic” means a moiety wherein theconstituent atoms make up an unsaturated ring system, all atoms in thering system are sp² hybridized and the total number of pi electrons isequal to 4n+2. An aromatic ring can be such that the ring atoms are onlycarbon atoms (e.g., aryl) or can include carbon and non-carbon atoms(e.g., heteroaryl).

As used herein, the term “substituted” refers to independent replacementof one or more (typically 1, 2, 3, 4, or 5) of the hydrogen atoms on thesubstituted moiety with substituents independently selected from thegroup of substituents listed below in the definition for “substituents”or otherwise specified. In general, a non-hydrogen substituent can beany substituent that can be bound to an atom of the given moiety that isspecified to be substituted. Examples of substituents include, but arenot limited to, acyl, acylamino, acyloxy, aldehyde, alicyclic,aliphatic, alkanesulfonamido, alkanesulfonyl, alkaryl, alkenyl, alkoxy,alkoxycarbonyl, alkyl, alkylamino, alkylcarbanoyl, alkylene, alkylidene,alkylthios, alkynyl, amide, amido, amino, amino, aminoalkyl, aralkyl,aralkylsulfonamido, arenesulfonamido, arenesulfonyl, aromatic, aryl,arylamino, arylcarbanoyl, aryloxy, azido, carbamoyl, carbonyl, carbonyls(including ketones, carboxy, carboxylates, CF₃, cyano (CN), cycloalkyl,cycloalkylene, ester, ether, haloalkyl, halogen, halogen, heteroaryl,heterocyclyl, hydroxy, hydroxy, hydroxyalkyl, imino, iminoketone,ketone, mercapto, nitro, oxaalkyl, oxo, oxoalkyl, phosphoryl (includingphosphonate and phosphinate), silyl groups, sulfonamido, sulfonyl(including sulfate, sulfamoyl and sulfonate), thiols, and ureidomoieties, each of which may optionally also be substituted orunsubstituted. In some cases, two substituents, together with thecarbon(s) to which they are attached to, can form a ring.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, and —O-alkynyl. Aroxy can berepresented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl areas defined below. The alkoxy and aroxy groups can be substituted asdescribed above for alkyl.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, and—S-alkynyl. Representative alkylthio groups include methylthio,ethylthio, and the like. The term “alkylthio” also encompassescycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.“Arylthio” refers to aryl or heteroaryl groups.

The term “sulfinyl” means the radical —SO—. It is noted that thesulfinyl radical can be further substituted with a variety ofsubstituents to form different sulfinyl groups including sulfinic acids,sulfinamides, sulfinyl esters, sulfoxides, and the like.

The term “sulfonyl” means the radical —SO₂—. It is noted that thesulfonyl radical can be further substituted with a variety ofsubstituents to form different sulfonyl groups including sulfonic acids,sulfonamides, sulfonate esters, sulfones, and the like.

The term “thiocarbonyl” means the radical —C(S)—. It is noted that thethiocarbonyl radical can be further substituted with a variety ofsubstituents to form different thiocarbonyl groups including thioacids,thioamides, thioesters, thioketones, and the like.

As used herein, the term “amino” means —NH₂. The term “alkylamino” meansa nitrogen moiety having at least one straight or branched unsaturatedaliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen.For example, representative amino groups include —NH₂, —NHCH₃, —N(CH₃)₂,—NH(C₁-C₁₀alkyl), —N(C₁-C₁₀alkyl)₂, and the like. The term “alkylamino”includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and“heterocyclylamino.” The term “arylamino” means a nitrogen moiety havingat least one aryl radical attached to the nitrogen. For example —NHaryl,and —N(aryl)₂. The term “heteroarylamino” means a nitrogen moiety havingat least one heteroaryl radical attached to the nitrogen. For example—NHheteroaryl, and —N(heteroaryl)₂. Optionally, two substituentstogether with the nitrogen can also form a ring. Unless indicatedotherwise, the compounds described herein containing amino moieties caninclude protected derivatives thereof. Suitable protecting groups foramino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl,and the like.

The term “aminoalkyl” means an alkyl, alkenyl, and alkynyl as definedabove, except where one or more substituted or unsubstituted nitrogenatoms (—N—) are positioned between carbon atoms of the alkyl, alkenyl,or alkynyl. For example, an (C₂-C₆) aminoalkyl refers to a chaincomprising between 2 and 6 carbons and one or more nitrogen atomspositioned between the carbon atoms.

It is noted in regard to all of the definitions provided herein that thedefinitions should be interpreted as being open ended in the sense thatfurther substituents beyond those specified may be included. Hence, a C₁alkyl indicates that there is one carbon atom but does not indicate whatare the substituents on the carbon atom. Hence, a C₁ alkyl comprisesmethyl (i.e., —CH3) as well as —CR_(a)R_(b)R_(c) where R_(a), R_(b), andR_(c) can each independently be hydrogen or any other substituent wherethe atom alpha to the carbon is a heteroatom or cyano. Hence, CF₃, CH₂OHand CH₂CN are all C₁ alkyls.

The term “derivative” as used herein refers to a chemical substancerelated structurally to another, i.e., an “original” substance, whichcan be referred to as a “parent” compound. A “derivative” can be madefrom the structurally-related parent compound in one or more steps. Insome embodiments, the general physical and chemical properties of aderivative can be similar to or different from the parent compound.

As used herein, the term “polyamine” refers to molecules comprising twoor more amines. Polyamines can be aliphatic, straight-chain aminesderived biosynthetically from amino acids. Several polyamines arereviewed in Marton et al. (1995) Ann. Rev. Pharm. Toxicol. 35:55-91,content of which is incorporated herein by reference. Polyaminescadaverine and putrescine are diamines produced by decarboxylation oflysine or ornithine, respectively. Putrescine is converted tospermidine, and spermidine to spermine, by the addition of anaminopropyl group. This group is provided by decarboxylated S-adenosylmethionine. “Polyamine analog” is defined as an organic cationstructurally similar but non-identical to polyamines such as spermineand/or spermidine and their precursor, diamine putrescine. Polyamineanalogs, which can be branched or unbranched, include, but are notlimited to, BE-4444 [1,19bis(ethylamino)-5,10,15-triazanonadecane];BE-333 [N1, N11-diethylnorspermine; DENSPM;1,11-bis(ethylamino)4,8-diazaundecane; thermine; Warner-Parke-Davis];BE-33 [N1,N7-bis (ethyl) norspermidine]; BE-34 [N1,N8-bis (ethyl)spermidine]; BE-44 [N1,N9-bis (ethyl) homospermidine]; BE-343[N1,N12-bis (ethyl) spermine; diethylspermine-N1-N12; DESPM]; BE-373[N,N-bis(3-ethylamino) propyl)-1,7-heptane diamine, Merrell-Dow]; BE-444[N1, N14-bis (ethyl) homospermine; diefhylhomospermine-N1N14]; BE-3443[1,17-bis (ethylamino)-4,9,14-triazaheptadecane]; BE-4334 [1,17-bis(ethylamino)-5,9,13triazaheptadecane]; 1,1 2-Me2-SPM[1,12dimethylspermine]; and the various polyamine analogs disclosed inWO 98/17624; U.S. Pat. No. 5,889,061; WO 00/66175 and WO 00/66587; andO'Sullivan et al. (1997) Bioorg. Med. Chem. 5:2145-2155 andMukhopadhyaya et al. (1995) Exp. Parasit. 81:39-46; and U.S. Pat. No.4,935,449, content of all of which is incorporated herein by reference.Cyclic polyamine compounds and cyclic polyamine analogs are disclosed inInternational Patent Application WO 02/10142. In certain of these cyclicpolyamine compounds, one or more of the aliphatic nitrogens form part ofan amide group

In some embodiments, the compounds described herein can be in the formof a prodrug. The term “prodrug” as used herein refers to compounds thatcan be converted via some chemical or physiological process (e.g.,enzymatic processes and metabolic hydrolysis) to compound describedherein. Thus, the term “prodrug” also refers to a precursor of abiologically active compound that is pharmaceutically acceptable. Aprodrug can be inactive when administered to a subject, i.e. an ester,but is converted in vivo to an active compound, for example, byhydrolysis to the free carboxylic acid or free hydroxyl. The prodrugcompound often offers advantages of solubility, tissue compatibility ordelayed release in an organism. The term “prodrug” is also meant toinclude any covalently bonded carriers, which release the activecompound in vivo when such prodrug is administered to a subject.Prodrugs of an active compound, as described herein, may be prepared bymodifying functional groups present in the active compound in such a waythat the modifications are cleaved, either in routine manipulation or invivo, to the parent active compound. Prodrugs include compounds whereina hydroxy, amino or mercapto group is bonded to any group that, when theprodrug of the active compound is administered to a subject, cleaves toform a free hydroxy, free amino or free mercapto group, respectively.For example, a compound comprising a hydroxy group can be administeredas an ester that is converted by hydrolysis in vivo to the hydroxycompound. Suitable esters that can be converted in vivo into hydroxycompounds include acetates, citrates, lactates, tartrates, malonates,oxalates, salicylates, propionates, succinates, fumarates, formates,benzoates, maleates, methylene-bis-b-hydroxynaphthoates, gentisates,isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group can be administered asan amide, e.g., acetamide, formamide and benzamide that is converted byhydrolysis in vivo to the amine compound. See Harper, “DrugLatentiation” in Jucker, ed. Progress in Drug Research 4:221-294 (1962);Morozowich et al, “Application of Physical Organic Principles to ProdrugDesign” in E. B. Roche ed. Design of Biopharmaceutical Propertiesthrough Prodrugs and Analogs, APHA Acad. Pharm. Sci. 40 (1977);Bioreversible Carriers in Drug in Drug Design, Theory and Application,E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987); Design of Prodrugs, H.Bundgaard, Elsevier (1985); Wang et al. “Prodrug approaches to theimproved delivery of peptide drug” in Curr. Pharm. Design. 5(4):265-287(1999); Pauletti et al. (1997) Improvement in peptide bioavailability:Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev.27:235-256; Mizen et al. (1998) “The Use of Esters as Prodrugs for OralDelivery of (3-Lactam antibiotics,” Pharm. Biotech. 11, 345-365;Gaignault et al. (1996) “Designing Prodrugs and Bioprecursors I. CarrierProdrugs,” Pract. Med. Chem. 671-696; Asgharnejad, “Improving Oral DrugTransport”, in Transport Processes in Pharmaceutical Systems, G. L.Amidon, P. I. Lee and E. M. Topp, Eds., Marcell Dekker, p. 185-218(2000); Balant et al., “Prodrugs for the improvement of drug absorptionvia different routes of administration”, Eur. J. Drug Metab.Pharmacokinet., 15(2): 143-53 (1990); Balimane and Sinko, “Involvementof multiple transporters in the oral absorption of nucleosideanalogues”, Adv. Drug Delivery Rev., 39(1-3): 183-209 (1999); Browne,“Fosphenytoin (Cerebyx)”, Clin. Neuropharmacol. 20(1): 1-12 (1997);Bundgaard, “Bioreversible derivatization of drugs—principle andapplicability to improve the therapeutic effects of drugs”, Arch. Pharm.Chemi 86(1): 1-39 (1979); Bundgaard H. “Improved drug delivery by theprodrug approach”, Controlled Drug Delivery 17: 179-96 (1987); BundgaardH. “Prodrugs as a means to improve the delivery of peptide drugs”, Arfv.Drug Delivery Rev. 8(1): 1-38 (1992); Fleisher et al. “Improved oraldrug delivery: solubility limitations overcome by the use of prodrugs”,Arfv. Drug Delivery Rev. 19(2): 115-130 (1996); Fleisher et al. “Designof prodrugs for improved gastrointestinal absorption by intestinalenzyme targeting”, Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A):360-81, (1985); Farquhar D, et al., “Biologically ReversiblePhosphate-Protective Groups”, Pharm. Sci., 72(3): 324-325 (1983);Freeman S, et al., “Bioreversible Protection for the Phospho Group:Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)Methylphosphonate with Carboxyesterase,” Chem. Soc., Chem. Commun.,875-877 (1991); Friis and Bundgaard, “Prodrugs of phosphates andphosphonates: Novel lipophilic alphaacyloxyalkyl ester derivatives ofphosphate- or phosphonate containing drugs masking the negative chargesof these groups”, Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangwar et al.,“Pro-drug, molecular structure and percutaneous delivery”, Des.Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21.(1977); Nathwani and Wood, “Penicillins: a current review of theirclinical pharmacology and therapeutic use”, Drugs 45(6): 866-94 (1993);Sinhababu and Thakker, “Prodrugs of anticancer agents”, Adv. DrugDelivery Rev. 19(2): 241-273 (1996); Stella et al., “Prodrugs. Do theyhave advantages in clinical practice?”, Drugs 29(5): 455-73 (1985); Tanet al. “Development and optimization of anti-HIV nucleoside analogs andprodrugs: A review of their cellular pharmacology, structure-activityrelationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1-3):117-151 (1999); Taylor, “Improved passive oral drug delivery viaprodrugs”, Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino andBorchardt, “Prodrug strategies to enhance the intestinal absorption ofpeptides”, Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus,“Concepts for the design of anti-HIV nucleoside prodrugs for treatingcephalic HIV infection”, Adv. Drug Delivery Rev.: 39(1-3):63-80 (1999);Waller et al., “Prodrugs”, Br. J. Clin. Pharmac. 28: 497-507 (1989),content of all of which are herein incorporated by reference in itsentirety.

The term “protected derivatives” means derivatives of compoundsdescribed herein in which a reactive site or sites are blocked withprotecting groups. Protected derivatives are useful in the preparationof compounds or in themselves can be active. A comprehensive list ofsuitable protecting groups can be found in T. W. Greene, ProtectingGroups in Organic Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999.

“Isomers” mean any compound having identical molecular formulae butdiffering in the nature or sequence of bonding of their atoms or in thearrangement of their atoms in space. Isomers that differ in thearrangement of their atoms in space are termed “stereoisomers”.Stereoisomers that are not mirror images of one another are termed“diastereomers” and stereoisomers that are nonsuperimposable mirrorimages are termed “enantiomers” or sometimes “optical isomers”. A carbonatom bonded to four nonidentical substituents is termed a “chiralcenter”. A compound with one chiral center has two enantiomeric forms ofopposite chirality. A mixture of the two enantiomeric forms is termed a“racemic mixture”. A compound that has more than one chiral center has2¹¹⁻¹ enantiomeric pairs, where n is the number of chiral centers.Compounds with more than one chiral center may exist as ether anindividual diastereomers or as a mixture of diastereomers, termed a“diastereomeric mixture”. When one chiral center is present astereoisomer may be characterized by the absolute configuration of thatchiral center. Absolute configuration refers to the arrangement in spaceof the substituents attached to the chiral center. Enantiomers arecharacterized by the absolute configuration of their chiral centers anddescribed by the R- and S-sequencing rules of Cahn, Ingold and Prelog.Conventions for stereochemical nomenclature, methods for thedetermination of stereochemistry and the separation of stereoisomers arewell known in the art (e.g., see “Advanced Organic Chemistry”, 4thedition, March, Jerry, John Wiley & Sons, New York, 1992).

The term “enantiomer” is used to describe one of a pair of molecularisomers which are mirror images of each other and non-superimposable.Other terms used to designate or refer to enantiomers include“stereoisomers” (because of the different arrangement or stereochemistryaround the chiral center; although all enantiomers are stereoisomers,not all stereoisomers are enantiomers) or “optical isomers” (because ofthe optical activity of pure enantiomers, which is the ability ofdifferent pure enantiomers to rotate planepolarized light in differentdirections). Enantiomers generally have identical physical properties,such as melting points and boiling points, and also have identicalspectroscopic properties. Enantiomers can differ from each other withrespect to their interaction with plane-polarized light and with respectto biological activity.

The designations “R” and “S” are used to denote the absoluteconfiguration of the molecule about its chiral center(s). Thedesignations may appear as a prefix or as a suffix; they may or may notbe separated from the isomer by a hyphen; they may or may not behyphenated; and they may or may not be surrounded by parentheses.

The designations or prefixes “(+)” and “(−)” are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)meaning that the compound is levorotatory (rotates to the left). Acompound prefixed with (+) is dextrorotatory (rotates to the right).

The term “racemic mixture,” “racemic compound” or “racemate” refers to amixture of the two enantiomers of one compound. An ideal racemic mixtureis one wherein there is a 50:50 mixture of both enantiomers of acompound such that the optical rotation of the (+) enantiomer cancelsout the optical rotation of the (−) enantiomer.

The term “resolving” or “resolution” when used in reference to a racemicmixture refers to the separation of a racemate into its twoenantiomorphic forms (i.e., (+) and (−); 65 (R) and (S) forms). Theterms can also refer to enantioselective conversion of one isomer of aracemate to a product.

The term “enantiomeric excess” or “ee” refers to a reaction productwherein one enantiomer is produced in excess of the other, and isdefined for a mixture of (+)- and (−)-enantiomers, with compositiongiven as the mole or weight or volume fraction F(+) and F(−) (where thesum of F(+) and F(−)=1). The enantiomeric excess is defined as *F(+)−F(−)* and the percent enantiomeric excess by 100x*F(+)−F(−)*. The“purity” of an enantiomer is described by its ee or percent ee value (%ee).

Whether expressed as a “purified enantiomer” or a “pure enantiomer” or a“resolved enantiomer” or “a compound in enantiomeric excess”, the termsare meant to indicate that the amount of one enantiomer exceeds theamount of the other. Thus, when referring to an enantiomer preparation,both (or either) of the percent of the major enantiomer (e.g. by mole orby weight or by volume) and (or) the percent enantiomeric excess of themajor enantiomer may be used to determine whether the preparationrepresents a purified enantiomer preparation.

The term “enantiomeric purity” or “enantiomer purity” of an isomerrefers to a qualitative or quantitative measure of the purifiedenantiomer; typically, the measurement is expressed on the basis of eeor enantiomeric excess.

The terms “substantially purified enantiomer,” “substantially resolvedenantiomer” “substantially purified enantiomer preparation” are meant toindicate a preparation (e.g. derived from non-optically active startingmaterial, substrate, or intermediate) wherein one enantiomer has beenenriched over the other, and more preferably, wherein the otherenantiomer represents less than 20%, more preferably less than 10%, andmore preferably less than 5%, and still more preferably, less than 2% ofthe enantiomer or enantiomer preparation.

The terms “purified enantiomer,” “resolved enantiomer” and “purifiedenantiomer preparation” are meant to indicate a preparation (e.g.derived from non-optically active starting material, substrates orintermediates) wherein one enantiomer (for example, the R-enantiomer) isenriched over the other, and more preferably, wherein the otherenantiomer (for example the S-enantiomer) represents less than 30%,preferably less than 20%, more preferably less than 10% (e.g. in thisparticular instance, the R-enantiomer is substantially free of theS-enantiomer), and more preferably less than 5% and still morepreferably, less than 2% of the preparation. A purified enantiomer maybe synthesized substantially free of the other enantiomer, or a purifiedenantiomer may be synthesized in a stereo-preferred procedure, followedby separation steps, or a purified enantiomer may be derived from aracemic mixture.

The term “enantioselectivity,” also called the enantiomeric ratioindicated by the symbol “E,” refers to the selective capacity of anenzyme to generate from a racemic substrate one enantiomer relative tothe other in a product racemic mixture; in other words, it is a measureof the ability of the enzyme to distinguish between enantiomers. Anonselective reaction has an E of 1, while resolutions with E's above 20are generally considered useful for synthesis or resolution. Theenantioselectivity resides in a difference in conversion rates betweenthe enantiomers in question. Reaction products are obtained that areenriched in one of the enantiomers; conversely, remaining substrates areenriched in the other enantiomer. For practical purposes it is generallydesirable for one of the enantiomers to be obtained in large excess.This is achieved by terminating the conversion process at a certaindegree of conversion.

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment. “Lesseffective” means that the treatment results in a therapeuticallysignificant lower level of pharmacological effectiveness and/or atherapeutically greater level of adverse physiological effects. Thecompounds described herein are effective in treating various types ofcancer.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount, for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means at least two standarddeviation (2SD) away from a reference level. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments of the aspects describedherein, the subject is a mammal, e.g., a primate, e.g., a human. Theterms, “patient” and “subject” are used interchangeably herein. Theterms, “patient” and “subject” are used interchangeably herein. Asubject can be male or female.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of humandiseases and disorders. In addition, compounds, compositions and methodsdescribed herein can be used to treat domesticated animals and/or pets.

In jurisdictions that forbid the patenting of methods that are practicedon the human body, the meaning of “administering” of a composition to ahuman subject shall be restricted to prescribing a controlled substancethat a human subject will self-administer by any technique (e.g.,orally, inhalation, topical application, injection, insertion, etc.).The broadest reasonable interpretation that is consistent with laws orregulations defining patentable subject matter is intended. Injurisdictions that do not forbid the patenting of methods that arepracticed on the human body, the “administering” of compositionsincludes both methods practiced on the human body and also the foregoingactivities.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. A compound or composition describedherein can be administered by any appropriate route known in the artincluding, but not limited to, oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),pulmonary, nasal, rectal, and topical (including buccal and sublingual)administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

Administration can also be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the compounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compounddescribed herein which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in a subject ata reasonable benefit/risk ratio applicable to any medical treatment.Thus, “therapeutically effective amount” means that amount which, whenadministered to a subject for treating a disease, is sufficient toeffect such treatment for the disease.

Determination of an effective amount is well within the capability ofthose skilled in the art. Generally, the actual effective amount canvary with the specific compound, the use or application technique, thedesired effect, the duration of the effect and side effects, thesubject's history, age, condition, sex, as well as the severity and typeof the medical condition in the subject, and administration of otherpharmaceutically active agents. Accordingly, an effective dose ofcompound described herein is an amount sufficient to produce at leastsome desired therapeutic effect in a subject.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of use or administration utilized.

The effective dose can be estimated initially from cell culture assays.A dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC₅₀ (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmacan be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay. The effective plasma concentration for a compound as disclosedherein can be about 0.01 μM to about 10 μM, about 0.2 μM to about 5 μM,or about 0.8 to about 3 μM in a subject, such as a rat, dog, or human.

Generally, the compositions are administered so that a compound of thedisclosure herein is used or given at a dose from 1 μg/kg to 1000 mg/kg;1 μg/kg to 500 mg/kg; 1 μg/kg to 150 mg/kg, 1 μg/kg to 100 mg/kg, 1μg/kg to 50 mg/kg, 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to1 mg/kg, 100 μg/kg to 100 mg/kg, 100 μg/kg to 50 mg/kg, 100 μg/kg to 20mg/kg, 100 μg/kg to 10 mg/kg, 100 μg/kg to 1 mg/kg, 1 mg/kg to 100mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It isto be understood that ranges given here include all intermediate ranges,for example, the range 1 mg/kg to 10 mg/kg includes 1 mg/kg to 2 mg/kg,1 mg/kg to 3 mg/kg, 1 mg/kg to 4 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6mg/kg, 1 mg/kg to 7 mg/kg, 1 mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to10 mg/kg, 6 mg/kg to 10 mg/kg, 7 mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg,9 mg/kg to 10 mg/kg, and the like. Further contemplated is a dose(either as a bolus or continuous infusion) of about 0.1 mg/kg to about10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, or 0.5 mg/kg to about 3mg/kg. It is to be further understood that the ranges intermediate tothose given above are also within the scope of this disclosure, forexample, in the range 1 mg/kg to 10 mg/kg, for example use or doseranges such as 2 mg/kg to 8 mg/kg, 3 mg/kg to 7 mg/kg, 4 mg/kg to 6mg/kg, and the like.

The compounds described herein can be administered at once, or can bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment will be a function of the location of where the composition isparenterally administered, the carrier and other variables that can bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values can also vary with the age of theindividual treated. It is to be further understood that for anyparticular subject, specific dosage regimens can need to be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of theformulations. Hence, the concentration ranges set forth herein areintended to be exemplary and are not intended to limit the scope orpractice of the claimed formulations.

The compound can be administered as a single bolus or multiple boluses,as a continuous infusion, or a combination thereof. For example, thecompound can be administered as a single bolus initially, and thenadministered as a continuous infusion following the bolus. The rate ofthe infusion can be any rate sufficient to maintain effective plasmaconcentration. Some contemplated infusion rates include from 1 μg/kg/minto 100 mg/kg/min, or from 1 μg/kg/hr to 1000 mg/kg/hr. Rates of infusioncan include 0.2 to 1.5 mg/kg/min, or more specifically 0.25 to 1mg/kg/min, or even more specifically 0.25 to 0.5 mg/kg/min. It will beappreciated that the rate of infusion can be determined based upon thedose necessary to maintain effective plasma concentration and the rateof elimination of the compound, such that the compound is administeredvia infusion at a rate sufficient to safely maintain a sufficienteffective plasma concentration of compound in the bloodstream.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

Generally, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent to a patient, orapplication or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, said patient having a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. Thus, treating can include suppressing,inhibiting, preventing, treating, or a combination thereof. Treatingrefers, inter alia, to increasing time to sustained progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof “Suppressing” or “inhibiting”,refers, inter alia, to delaying the onset of symptoms, preventingrelapse to a disease, decreasing the number or frequency of relapseepisodes, increasing latency between symptomatic episodes, reducing theseverity of symptoms, reducing the severity of an acute episode,reducing the number of symptoms, reducing the incidence ofdisease-related symptoms, reducing the latency of symptoms, amelioratingsymptoms, reducing secondary symptoms, reducing secondary infections,prolonging patient survival, or a combination thereof. In one embodimentthe symptoms are primary, while in another embodiment symptoms aresecondary. “Primary” refers to a symptom that is a direct result of adisorder, e.g., diabetes, while, secondary refers to a symptom that isderived from or consequent to a primary cause. Symptoms may be anymanifestation of a disease or pathological condition.

Accordingly, as used herein, the term “treatment” or “treating” includesany administration of a compound described herein and includes: (i)preventing the disease from occurring in a subject which may bepredisposed to the disease but does not yet experience or display thepathology or symptomatology of the disease; (ii) inhibiting the diseasein an subject that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., arresting further development ofthe pathology and/or symptomatology); or (iii) ameliorating the diseasein a subject that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., reversing the pathology and/orsymptomatology).

By “treatment”, “prevention” or “amelioration” of a disease or disorderis meant delaying or preventing the onset of such a disease or disorder,reversing, alleviating, ameliorating, inhibiting, slowing down orstopping the progression, aggravation or deterioration the progressionor severity of a condition associated with such a disease or disorder.In one embodiment, the symptoms of a disease or disorder are alleviatedby at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,or at least 50%.

Specifically, by “treatment, prevention or amelioration ofneurodegenerative disorder” is meant delaying or preventing the onset ofsuch a disorder (e.g. death of motor neurons), at reversing,alleviating, ameliorating, inhibiting, slowing down or stopping theprogression, aggravation or deterioration the progression or severity ofsuch a condition. In one embodiment, the symptom of a neurodegenerativedisorder is alleviated by at least 20%, at least 30%, at least 40%, orat least 50%. In one embodiment, the symptom of a neurodegenerativedisease is alleviated by more than 50%. In one embodiment, the symptomof a neurodegenerative disorder is alleviated by 80%, 90%, or greater.

Efficacy of treatment is determined in association with any known methodfor diagnosing the disorder. Alleviation of one or more symptoms of thedisorder indicates that the compound confers a clinical benefit. Any ofthe therapeutic methods described to above can be applied to anysuitable subject including, for example, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and most preferably, humans.

By “embryonic stem cell” is meant a cell, derived from an embryo at theblastocyst stage, or before substantial differentiation of the cell intothe three germ layers, that can self renew and displays morphologicalcharacteristics of undifferentiated cells, distinguishing them fromdifferentiated cells of embryonic or adult origin. Exemplarymorphological characteristics include high nuclear/cytoplasmic ratiosand prominent nucleoli under a microscope. Under appropriate conditionsknown to the skilled artisan, embryonic stem cells can differentiateinto cells or tissues of the three germ layers: endoderm, mesoderm, andectoderm. Assays for identification of an embryonic stem cell includethe ability to form a teratoma in a suitable host or to be stained formarkers of an undifferentiated cell such as Oct-4.

By “an amount sufficient to treat” is meant the amount of a compoundrequired to improve, inhibit, or ameliorate a condition of a subject, ora symptom of a disease, in a clinically relevant manner. Any improvementin the subject is considered sufficient to achieve treatment.

By “Nurr1 biological activity” is meant any activity known to be causedin vitro, in vivo or ex vivo by a Nurr1 polypeptide. For example, suchactivity could include activating transcription of tyrosine hydroxylase.

“Nurr1 nucleic acid” and “Nurr1 gene” are used interchangeably hereinand refer to a nucleic acid that encodes all or a portion of a Nurr1polypeptide, or is substantially identical to all or a portion of thenucleic acid sequence of Genbank Accession No. ABO1 7586 (Ichinose etal., Gene 230:233-239, 1999), or analog thereof. By “Nurr1 polypeptide”is meant a polypeptide substantially identical to all or a portion ofthe polypeptide sequence of Genbank Accession No. BAA75666, or analogthereof, and having Nurr1 biological activity.

EXAMPLES Example 1 Synthetic Protocol (Representative Examples)

Among the prototype structures of FIG. 1, synthesis of4-aminoquinoline-pyrimidine hybrids is described here. Similarly, othercompounds as depicted in FIG. 1 were also synthesized. The4-aminoquinoline-pyrimidine conjugates were synthesized using three stepprocedure as outlined in Scheme 1. The commercially available startingmaterial 4,7-dichloroquinoline (8) was reacted with an excess ofaliphatic linear chain diaminoalkanes via SNAr type of reaction in neatconditions as reported in literature to afford substituted4-aminoquinolines (9a-d) with free terminal amino group in good toexcellent yield [Roepe, P. D. J. Med. Chem. 2008, 51, 3466]. Theseintermediates (9a-d) on reaction with commercially available2,4-dichloro-6-methyl-pyrimidine yielded two regioisomers viz 10a-d inmajor and 11a-d in minor yield. The major regioisomers 10a-d wassubjected to nucleophilic substitution with different cyclic secondaryamines at an elevated temperature in DMF as solvent, to yield4-aminoquinoline-pyrimidine conjugates (12a-n) in excellent yield.

In vitro antimalarial activity of all the conjugates was studied againstboth CQ-sensitive (D6 clone) and CQ-resistant (W2 clone) strains of P.falciparum while mammalian cell cytotoxicity was determined againstVero, LLC-PK-1, HepG2 cells (Table 6) using procedure as describedearlier [Jain, R. Bioorg. Med. Chem. 2005, 13, 4458; Khan, I. A. Lipids2004, 37, 169]. Most of the compounds have shown very promisingantimalarial activity. Out of these, eleven compounds (12b-c, 12e-f,12h-n) have displayed better antimalarial activity (IC50=0.006 μM to0.041 μM) against CQ-sensitive strain, while thirteen compounds (10a,12b-f, 12h-n) have displayed better antimalarial activity (IC50=0.016 μMto 0.34 μM) against CQ-resistant strains of P. falciparum. Theselectivity index of antimalarial activity (calculated as a ratio ofIC50 for cytotoxicity to Vero cells and IC50 for antimalarial activity)was very high for most of these compounds as compared to standard drugchloroquine (CQ). The activity of (12i, 12j, 12l and 12m) was 7-8 foldhigher than CQ and 2 fold higher than artemisinin in CQ-sensitive strainrevealing their strong potency. The comparison of antimalarial activityof 10a-d with 12a-n clearly showed that substitution of Cl fromcompounds 10a-d (IC50=0.15 μM to 0.46 μM for CQ-sensitive and 0.34 μM to0.74 μM for CQ-resistant) with secondary amines (12b-f and 12h-n,IC50=0.006 μM to 0.066 μM for CQ-sensitive and 0.016 μM to 0.211 μM forCQ-resistant) improves antimalarial activity of these compounds. Thecomparison of antimalarial activity of two groups of regioisomers 10a-d(IC50=0.15 μM to 0.46 μM for CQ-sensitive and 0.34 μM to 0.74 μM forCQ-resistant) and 11a-d (IC50=0.16 μM to 0.27 μM for CQ-sensitive and0.56 μM to 0.1.24 μM for CQ-resistant) clearly indicates that both theregioisomers displayed more or less similar potency against both thestrains of P. falciparum. These results indicate that the point ofattachment of the spacer to the pyrimidine nucleus may not have a greatimpact on activity profile. For a particular amino substituted4-aminoquinoine-pyrimidine hybrids (12a-d or 12e-h or 12i-l), structureactivity relationship (SAR) study demonstrated no obvious trend ofactivity with increasing or decreasing carbon spacer from C2 to C6 butchanging the amino groups for a particular C2 (12e, 12i), C3 (12b, 12f,12j, 12m), C4 (12c, 12k, 12n) or C6 (12d, 12h, 12l ) spacer changes theactivity significantly in a decreasing order of 4-ethylpiperazine>4-methyl piperazine>morpholine>piperidine.

Among the most active compounds (12b-12f, 12h-12n), although 12d, 12h,and 12l showed toxicity to all the three cell lines (VERO, LLC-PK-1, andHepG2) but their selectivity index of antimalarial activity wasconsiderably high (Table 7). In general, the cytotoxicity of the most ofthe conjugates was at much higher concentrations than the concentrationsresponsible for their antimalarial activity. Some of the hybrids werenot cytotoxic at all up to the highest tested concentration of 60 μMindicating their high selectivity index of antimalarial activity versuscytotoxicity to mammalian cells.

TABLE 6 In-vitro antimalarial activity of 4-aminoquinoline-pyrimidinehybrids P. falciparum P. falciparum Comp. (D6 Clone) (W2 Clone) NoStructure IC50 (μM) S.I. IC50 (μM) S.I. 10a

0.21 (>285) 0.34 (>176) 10b

0.35 (>170) 0.74 (>80) 10c

0.151 (>398) 0.690 (>87) 10d

0.469 73.7 0.544 27.2 11a

0.27 159.2 0.91 47.2 11b

0.27 (>222) 1.24 (>48) 11c

0.196 (>306) 0.664 (>90) 11d

0.165 336.9 0.568 97.8 12a

ND ND ND ND 12b

0.026 (>2300) 0.211 (>284) 12c

0.021 (>2857) 0.082 (>730) 12d

0.066 142.4 0.128 73.4 12e

0.025 (>2400) 0.12 (>500) 12f

0.022 (>2727) 0.058 (>1034) 12g

ND ND ND ND 12h

0.041 417.0 0.120 142.5 12i

0.006 (>10000) 0.046 (>1304) 12j

0.007 (>8571) 0.063 (>952) 12k

0.022 (>2727) 0.021 (>2857) 12l

0.007 (>1457) 0.016 (>637) 12m

0.006 (>10000) 0.061 (>983) 12n

0.019 1678.9 0.018 1772.2 Chloroquine 0.048 1250 0.43 140 Artemisinine0.012 5000 0.012 5000

TABLE 7 In vitro cytotoxicity of 4-aminoquinoline- pyrimidine hybrids tomammalian cells. IC50 (μM) Comp. No VERO LLC-PK-1 HepG2 10a NC NC NC 10bNC NC NC 10c NC NC NC 10d 34.6 14.8  9.3 11a 43.0 41.6 43.0 11b NC NC NC11c NC NC 39.8 11d 55.6 23.4  8.1 12a ND ND ND 12b NC NC NC 12c NCNC >60   12d  9.4 <6.7 <6.7 12e NC NC NC 12f NC 35.1 26.1 12g ND ND ND12h 17.1  9.8 <6.7 12i NC NC NC 12j NC 35.2 25.8 12k NC 34.0 27.2 12l10.2  7.4 <6.7 12m NC NC NC 12n 31.9 10.5 14.9 Doxorubicin  8.2  1.0<1.0

Considering the excellent in-vitro activity of these compounds, two ofthe hybrids 12i and 12m (Table 8) were subjected to in-vivo antimalarialactivity evaluation. The results showed 100% parasitemia suppression onday 5 for both the compounds (12i and 12m) with the survival rate of 5/5and 4/5 respectively on day 28 when the mice's are given a dose of 30mg/kg for 3 days post infection. No apparent toxicity is also observedfor these compounds. These results are found to be better than thestandard drug (Chloroquine) which gives 100% parasitemia suppression onday 5 when a 100 mg/kg dose was given for 3 days post infection.

These excellent results on animal models clearly depicted the potency ofthese compounds and give an insight about considering them for futuredrug candidates.

TABLE 8 In-vivo antimalarial activity of 4-aminoquinoline-pyrimidinehybrids 12i and 12m. Dose (mg/kg × % Parasitemia no. of dayssuppression¹ Cage Treatment post-infection) Day 5 Day 7 Survival² Day ofDeath MST³ Cure⁴ Remarks 1 Vehicle PO NA × 4  — — 0/5 11/11/12/21/2115.2 0/5 2 Chloroquine PO 100 × 3  100.00 100.00 5/5 28/28/28/28/28 280/5 3 12i PO 3.3 × 3  23.52 35.21 1/5 21/28/21/13/13 19.2 No apparent0/5 Toxicity 4 12i PO 10 × 3 97.03 2.03 0/5 21/22/22/21/21 21.4 Noapparent 0/5 Toxicity 5 12i PO 30 × 3 100.00 100.00 5/5 28/28/28/28/2828 No apparent 4/5 Toxicity 6 12m PO 3.3 × 3  20.12 19.09 0/517/21/21/21/21 20.2 No apparent 0/5 Toxicity 7 12m PO 10 × 3 73.89 27.140/5 7/21/20/7/21 15.2 No apparent 0/5 Toxicity 8 12m PO 30 × 3 100.00100.00 4/5 28/28/28/28/24 27.2 No apparent 1/5 Toxicity ¹1% suppressionin parasitemia is calculated by considering the mean marasitemia in thevehical control as 100%. Parasitemia suppression <80% is considered asnon-significant. ²Number of animals that survived day 28/total animalsin group (the day of the death-post-infection). ³MST—mean survival time(days). ⁴Number of mice without parasitemia (cured) till day 28post-infection. *Not included in analysisGeneral Procedure for the Synthesis of Selected Compounds (10a-d and11a-d):

To a well stirred solution of 2,4-dichloro-6-methyl-pyrimidine (2.0 g,12.2 mmol) and triethylamine (2.48 g, 24.5 mmol) in ethanol (50 ml) atroom temperature was added diamines 9a-d (12.2 mmol). The reactionmixture was allowed to stir overnight at room temperature. Aftercompletion of reaction as evident by TLC, reaction mixture was pouredinto ice cold water (250 ml) and precipitate thus formed was filteredand washed with excess of water at vacuum pump. The crude precipitatewas then dried, dissolved in 100 ml of CHCl3 and extracted with water(2×500 ml) and finally with brine. Excess of solvent was evaporated todryness under vacuum and the crude product thus obtained was purified bySiO2 column using MeOH/CHCl3 as eluent to yield respective compounds13a-d and 14a-d.

General Procedure for the Synthesis of Selected Compounds (12a-n)

In a 100 ml round bottom flask, compound 10a-d (1 eq.) was taken anddissolved in 10 ml of DMF. To this, a solution of respective amine (3eq.) in DMF (5 ml) was added dropwise. Reaction mixture was allowed tostir at 100-120° C. for 10 hours monitored by TLC. After completion,water (50 ml) was added to reaction mixture and it was extracted withEtOAc (2×25 ml). Organic layer was then collected, washed with water(2×100 ml) and brine, dried over Na2SO4 and finally excess of solventwas evaporated under vacuum. The crude residue thus obtained waspurified by SiO2 column using MeOH/CHCl3 as eluent to afford respectivecompounds 15a-n.

Spectral Data of Selected Compounds:

N-(7-chloro-quinolin-4-yl)-N′-(6-methyl-2-piperidin-1-yl-pyrimidin-4-yl)-ethane-1,2-diamine(12a): White solid. Yield: 85%; mp 177-179° C.; IR (cm-1, KBr): 3385,3344, 2941, 1580, 1447, 1331, 1237, 1141, 790; 1H NMR (400 MHz,DMSO-d6): 1.36-1.49 (m, 6H), 2.02 (s, 3H), 3.31-3.41 (m, 8H), 5.85 (s,1H), 6.56-6.72 (m, 2H), 7.34 (dd, 1H, J=11.0 Hz, 2.0 Hz), 7.41 (brs,1H), 7.68 (d, 1H, J=2.3 Hz); 8.07 (d, 1H, J=8.7 Hz), 8.28 (d, 1H, J=5.5Hz); ESI-MS (m/z): 397.22 (M+H)+; Anal. Calcd for C21H25ClN6: C, 63.55;H, 6.35; N, 21.17; Found: C, 63.53; H, 6.39; N, 21.26.

N-(7-Chloro-quinolin-4-yl)-N′-(6-methyl-2-piperidin-1-yl-pyrimidin-4-yl)-propane-1,3-diamine(12b): Pale yellow solid; Yield: 82%; mp 194-196° C.; IR (cm-1, KBr):3241, 3079, 1940, 1615, 1587, 1361, 1208, 1001, 804; 1H NMR (400 MHz,CDCl3): 1.46-1.56 (m, 6H), 1.87 (quin, 2H), 2.16 (s, 3H), 3.36 (q, 2H),4.45 (t, 4H), 3.49 (q, 2H), 4.86 (brs, 1H), 5.72 (s, 1H), 5.92 (brs,1H), 6.32 (d, 1H, J=5.4 Hz), 7.22 (dd, 1H, J=11.0 Hz, 2.0 Hz), 7.66 (d,1H, J=8.7 Hz), 7.85 (d, 1H, J=2.3 Hz), 8.41 (d, 1H, J=5.5 Hz); 13C NMR(100 MHz, CDCl3): 24.36, 24.67, 25.45, 29.06, 38.55, 40.34, 44.89,92.36, 98.86, 117.43, 121.55, 124.83, 128.53, 134.63, 149.18, 149.91,151.96, 162.42, 162.84, 165.62; ESI-MS (m/z): 411.23 (M+H)+; Anal. Calcdfor C22H27ClN6: C, 64.30; H, 6.62; N, 20.45; Found: C, 64.42; H, 6.68;N, 20.41.

N-(7-Chloro-quinolin-4-yl)-N′-(6-methyl-2-piperidin-1-yl-pyrimidin-4-yl)-butane-1,4-diamine(12c): White solid; Yield: 88%; mp 187-189° C.; IR (cm-1, KBr): 3247,3067, 2935, 1581, 1364, 1210, 1079, 848; 1H NMR (400 MHz, CDCl3):1.49-1.65 (m, 6H), 1.73-1.79 (m, 2H), 1.83-1.89 (m, 2H), 2.17 (s, 3H),3.35 (q, 2H), 3.45-3.52 (m, 6H), 4.79 (brs, 1H), 5.25 (brs, 1H), 5.76(s, 1H), 6.38 (d, 1H, J=5.5 Hz), 7.31 (dd, 1H, J=11.0 Hz, 2.0 Hz), 7.63(d, 1H, J=8.7 Hz), 7.93 (d, 1H, J=2.3 Hz), 8.50 (d, 1H, J=5.5 Hz); 13CNMR (100 MHz, CDCl3): 24.09, 24.67, 25.43, 25.75, 27.64, 40.64, 43.02,44.87, 91.91, 98.92, 117.14, 121.21, 125.04, 128.56, 134.66, 149.05,149.76, 151.93, 162.17, 162.90, 165.67; ESI-MS (m/z): 425.28 (M+H)+;Anal. Calcd for C23H29ClN6: C, 65.00; H, 6.88; N, 19.78; Found: C,64.98; H, 6.90; N, 19.81.

N-(7-Chloro-quinolin-4-yl)-N′-(6-methyl-2-piperidin-1-yl-pyrimidin-4-yl)-hexane-1,6-diamine(12d): Light brown solid; Yield: 82%; mp 97-99° C.; IR (cm-1, KBr):3314, 2933, 1579, 1368, 1232, 1134, 983, 853, 789; 1H NMR (400 MHz,CDCl3): 1.47-1.64 (m, 12H), 1.72-1.79 (m, 2H), 2.17 (s, 3H), 3.29 (q,2H), 3.39 (q, 2H), 3.54 (t, 4H), 4.76 (brs, 1H), 5.07 (brs, 1H), 5.74(s, 1H), 6.39 (d, 1H, J=5.5 Hz), 7.34 (dd, 1H, J=11.0 Hz, 2.0 Hz), 7.67(d, 1H, J=8.7 Hz), 7.95 (d, 1H, J=2.3 Hz), 8.51 (d, 1H, J=5.5 Hz);ESI-MS (m/z): 453.12 (M+H)+; Anal. Calcd for C25H33ClN6: C, 66.28; H,7.34; N, 18.55; Found: C, 66.32; H, 7.35; N, 18.49.

N-(7-Chloro-quinolin-4-yl)-N′-(6-methyl-2-morpholin-4-yl-pyrimidin-4-yl)-ethane-1,2-diamine(12e): Pale yellow solid; Yield: 86%; mp 165-167° C.; IR (cm-1, KBr):3391, 3245, 2954, 1585, 1438, 1228, 1110, 993; 1H NMR (400 MHz,DMSO-d6): 2.09 (s, 3H), 3.35-3.48 (m, 8H), 3.57 (t, 4H), 5.91 (s, 1H),6.62-6.70 (m, 2H), 7.41 (dd, 1H, J=11.0 Hz, 2.0 Hz), 7.44 (brs, 1H),7.74 (d, 1H, J=2.3 Hz), 8.14 (d, 1H, J=9.1 Hz), 8.35 (d, 1H, J=5.5 Hz);ESI-MS (m/z): 399.20 (M+H)+; Anal. Calcd for C20H23ClN6O: C, 60.22; H,5.81; N, 21.07; Found: C, 60.34; H, 5.79; N, 21.09.

N-(7-Chloro-quinolin-4-yl)-N′-(6-methyl-2-morpholin-4-yl-pyrimidin-4-yl)-propane-1,3-diamine(12f): White solid; Yield: 82%; mp 165-167° C.; IR (cm-1, KBr): 3233,3063, 2954, 1576, 1366, 1247, 1123, 791; 1H NMR (400 MHz, CDCl3): 1.92(quin, 2H), 2.20 (s, 3H), 3.39 (q, 2H), 3.47 (t, 4H), 3.52 (q, 2H), 3.66(t, 4H), 5.22 (brs, 1H), 5.71 (s, 1H), 5.95 (brs, 1H), 6.34 (d, 1H,J=5.4 Hz), 7.25 (dd, 1H, J=11.0 Hz, 2.2 Hz), 7.72 (d, 1H, J=8.7 Hz),7.89 (d, 1H, J=2.3 Hz), 8.44 (d, 1H, J=5.5 Hz); 13C NMR (100 MHz,CDCl3): 24.10, 28.85, 38.56, 40.34, 44.09, 66.46, 92.27, 98.85, 117.35,121.52, 124.93, 128.46, 134.73, 149.06, 149.90, 151.83, 161.87, 163.27,165.70; ESI-MS (m/z): 413.21 (M+H)+; Anal. Calcd for C21H25ClN6O: C,61.08; H, 6.10; N, 20.35; Found: C, 61.12; H, 6.17; N, 20.40.

N-(7-Chloro-quinolin-4-yl)-N′-(6-methyl-2-morpholin-4-yl-pyrimidin-4-yl)-butane-1,4-diamine(12g): White solid; Yield: 80%; mp 214-216° C.; IR (cm-1, KBr): 3256,3066, 2964, 1583, 1367, 1245, 1122, 994, 790; 1H NMR (400 MHz, CDCl3):1.72-1.80 (m, 2H), 1.83-1.91 (m, 2H), 2.20 (s, 3H), 3.36 (q, 2H), 3.48(q, 2H), 3.52 (t, 4H), 3.71 (t, 4H), 4.83 (brs, 1H), 5.19 (brs, 1H),5.75 (s, 1H), 6.40 (d, 1H, J=5.5 Hz), 7.32 (dd, 1H, J=11.0 Hz, 2.0 Hz),7.63 (d, 1H, J=8.7 Hz), 7.95 (d, 1H, J=2.3 Hz), 8.51 (d, 1H, J=5.5 Hz);13C NMR (100 MHz, CDCl3): 22.75, 24.18, 25.83, 41.39, 42.69, 64.93,89.96, 97.19, 122.10, 122.28, 122.80, 126.34, 132.68, 147.77, 149.11,150.43, 160.69, 161.99, 164.64; ESI-MS (m/z): 427.12 (M+H)+; Anal. Calcdfor C22H27ClN6O: C, 61.89; H, 6.37; N, 19.68; Found: C, 61.99; H, 6.45;N, 19.70.

N-(7-Chloro-quinolin-4-yl)-N′-(6-methyl-2-morpholin-4-yl-pyrimidin-4-yl)-hexane-1,6-diamine(12h): Pale yellow solid; Yield: 90%; mp 107-109° C.; IR (cm-1, KBr):3245, 2935, 2855, 1579, 1366, 1220, 1123, 994, 789; 1H NMR (400 MHz,CDCl3): 1.44-1.46 (m, 4H), 1.55-1.62 (m, 2H), 1.69-1.74 (m, 2H), 3.27(q, 2H), 3.36 (q, 2H), 3.54 (t, 4H), 3.78 (t, 4H), 4.88 (brs, 1H), 5.26(brs, 1H), 5.73 (s, 1H), 6.37 (d, 1H, J=5.1 Hz), 7.31 (dd, 1H, J=11.0Hz, 2.0 Hz), 7.70 (d, 1H, J=8.7 Hz), 7.93 (d, 1H, J=2.2 Hz), 8.50 (d,1H, J=5.1 Hz); 13C NMR (100 MHz, CDCl3): 24.13, 26.62, 26.82, 28.66,29.60, 41.04, 43.06, 44.09, 66.53, 91.58, 98.93, 117.07, 121.00, 125.08,128.62, 134.67, 149.04, 149.68, 151.94, 162.00, 163.45, 166.31; ESI-MS(m/z): 456.21 (M+H)+; Anal. Calcd for C24H31ClN6O: C, 63.35; H, 6.87; N,18.47; Found: C, 63.29; H, 6.91; N, 18.46.

N-(7-Chloro-quinolin-4-yl)-N′-[6-methyl-2-(4-methyl-piperazin-1-yl)-pyrimidin-4-yl]-ethane-1,2-diamine(12i): White solid 83%; Yield: mp 134-136° C.; IR (cm-1, KBr): 3385,3066, 2940, 1581, 1445, 1305, 1139, 997, 793; 1H NMR (400 MHz, CDCl3):2.29 (s, 3H), 2.30 (s, 3H), 2.42 (t, 4H), 3.41 (q, 2H), 3.62 (t, 4H),3.84 (q, 2H), 5.51 (brs, 1H), 5.87 (s, 1H), 6.30 (d, 1H, J=5.5 Hz), 6.90(brs, 1H), 7.21 (dd, 1H, J=11.0 Hz, 2.0 Hz), 7.55 (d, 1H, J=8.7 Hz),7.89 (d, 1H, J=2.3 Hz), 8.46 (d, 1H, J=5.5 Hz); ESI-MS (m/z): 412.26(M+H)+; Anal. Calcd for C21H26ClN7: C, 61.23; H, 6.36; N, 23.80; Found:C, 61.18; H, 6.51; N, 23.85.

N-(7-Chloro-quinolin-4-yl)-N′-[6-methyl-2-(4-methyl-piperazin-1-yl)-pyrimidin-4-yl]-propane-1,3-diamine(12j): Off white solid; Yield: 80%; mp 176-178° C.; IR (cm-1, KBr):3257, 3065, 2938, 1580, 1369, 1236, 1142, 1001, 789; 1H NMR (400 MHz,CDCl3): 1.94 (quin, 2H), 2.21 (s, 3H), 2.28 (s, 3H), 2.36 (t, 4H), 3.42(q, 2H), 3.52-3.56 (m, 6H), 5.32 (brs, 1H), 5.74 (s, 1H), 6.01 (brs,1H), 6.37 (d, 1H, J=5.4 Hz), 7.27 (dd, 1H, J=11.0 Hz, 2.0 Hz), 7.75 (d,1H, J=8.7 Hz), 7.91 (d, 1H, J=2.3 Hz), 8.46 (d, 1H, J=5.5 Hz); ESI-MS(m/z): 426.32 (M+H)+; Anal. Calcd for C22H28ClN7: C, 62.03; H, 6.63; N,23.02; Found: C, 62.11; H, 6.68; N, 22.95.

N-(7-Chloro-quinolin-4-yl)-N′-[6-methyl-2-(4-methyl-piperazin-1-yl)-pyrimidin-4-yl]-butane-1,4-diamine(12k): White solid; Yield: 87%; mp 183-185° C.; IR (cm-1, KBr): 3248,3072, 2927, 1581, 1367, 1280, 1139, 997, 790; 1H NMR (400 MHz, CDCl3):1.75-1.91 (m, 4H), 2.19 (s, 3H), 2.29 (s, 3H), 2.39 (t, 4H), 3.36 (q,2H), 3.48 (q, 2H), 3.56 (t, 4H), 4.86 (brs, 1H), 5.23 (brs, 1H), 5.77(s, 1H), 6.39 (d, 1H, J=5.5 Hz), 7.32 (dd, 1H, J=11.0 Hz, 2.0 Hz), 7.64(d, 1H, J=8.7 Hz), 7.94 (d, 1H, J=2.3 Hz), 8.51 (d, 1H, J=5.5 Hz); 13CNMR (100 MHz, CDCl3): 24.15, 25.79, 27.65, 40.65, 43.04, 43.64, 46.10,54.61, 92.00, 98.97, 117.12, 121.09, 125.09, 128.69, 134.67, 149.11,149.68, 151.99, 162.13, 163.16, 166.16; ESI-MS (m/z): 440.19 (M+H)+;Anal. Calcd for C23H30ClN7: C, 62.79; H, 6.87; N, 22.28; Found: C,62.80; H, 6.85; N, 22.30.

N-(7-Chloro-quinolin-4-yl)-N′-[6-methyl-2-(4-methyl-piperazin-1-yl)-pyrimidin-4-yl]-hexane-1,6-diamine(12l): Light brown solid; Yield: 85%; mp 152-154° C.; IR (cm-1, KBr):3266, 3068, 2931, 1580, 1367, 1279, 1164, 993, 789; 1H NMR (400 MHz,CDCl3): 1.46-1.55 (m, 4H), 1.59-1.61 (m, 2H), 1.74-1.77 (m, 2H), 2.19(s, 3H), 2.30 (s, 3H), 2.42-2.44 (m, 4H), 3.29 (q, 2H), 3.38 (q, 2H),3.60 (t, 4H), 4.77 (brs, 1H), 5.02 (brs, 1H), 5.75 (s, 1H), 6.40 (d, 1H,J=5.5 Hz), 7.35 (dd, 1H, J=11.0 Hz, 2.1 Hz), 7.66 (d, 1H, J=8.7 Hz),7.95 (d, 1H, J=2.3 Hz), 8.51 (d, 1H, J=5.5 Hz); 13C NMR (100 MHz,CDCl3): 24.14, 26.62, 26.82, 28.70, 29.63, 41.05, 43.10, 43.64, 46.12,54.67, 91.72, 98.98, 117.08, 120.96, 125.14, 128.70, 134.71, 149.07,149.67, 151.98, 162.05, 163.21, 166.09; ESI-MS (m/z): 468.32 (M+H)+;Anal. Calcd for C25H34ClN7: C, 64.15; H, 7.32; N, 20.95; Found: C,64.09; H, 7.31; N, 20.98.

N-(7-Chloro-quinolin-4-yl)-N′-[2-(4-ethyl-piperazin-1-yl)-6-methyl-pyrimidin-4-yl]-propane-1,3-diamine(12m): White solid; Yield: 82%; mp 184-186° C.; IR (cm-1, KBr): 3233,2967, 2812, 1579, 1366, 1250, 1132, 998; 1H NMR (400 MHz, CDCl3): 1.10(t, 3H), 1.95 (quin, 2H), 2.24 (s, 3H), 2.40-2.44 (m, 6H), 3.43 (q, 2H),3.55-3.59 (m, 6H), 4.94 (brs, 1H), 5.78 (s, 1H), 5.92 (brs, 1H), 6.40(d, 1H, J=5.4 Hz), 7.30 (dd, 1H, J=11.0 Hz, 2.0 Hz), 7.73 (d, 1H, J=8.7Hz), 7.93 (d, 1H, J=2.3 Hz), 8.49 (d, 1H, J=5.4 Hz); 13C NMR (100 MHz,CDCl3): 11.86, 24.38, 29.05, 38.50, 40.31, 43.64, 52.26, 52.34, 92.35,98.85, 117.38, 121.47, 124.84, 128.53, 134.61, 149.15, 149.84, 151.94,162.34, 163.04, 166.03; ESI-MS (m/z): 440.16 (M+H)+; Anal. Calcd forC23H30ClN7: C, 62.79; H, 6.87; N, 22.28; Found: C, 62.75; H, 6.89; N,22.20.

N-(7-Chloro-quinolin-4-yl)-N′-[2-(4-ethyl-piperazin-1-yl)-6-methyl-pyrimidin-4-yl]-butane-1,4-diamine(12n): White solid; Yield: 86%; mp 101-103° C.; IR (cm-1, KBr): 3255,2939, 2813, 1583, 1372, 1249, 1127, 994, 791; 1H NMR (400 MHz, CDCl3):1.09 (t, 3H), 1.73-1.80 (m, 2H), 1.84-1.91 (m, 2H), 2.19 (s, 3H),2.38-2.43 (m, 6H), 3.35 (q, 2H), 3.48 (q, 2H), 3.57 (t, 4H), 4.83 (brs,1H), 5.23 (brs, 1H), 5.77 (s, 1H), 6.39 (d, 1H, J=5.5 Hz), 7.31 (dd, 1H,J=11.0 Hz, 2.0 Hz), 7.64 (d, 1H, J=8.7 Hz), 7.94 (d, 1H, J=2.3 Hz), 8.51(d, 1H, J=5.5 Hz); ESI-MS (m/z): 454.11 (M+H)+; Anal. Calcd forC24H32ClN7: C, 63.49; H, 7.10; N, 21.60; Found: C, 63.59; H, 7.18; N,21.63.

Example 2: Agonists for Orphan Nuclear Receptor Nurr1 ExertNeuroprotective Effects and Rescue Behavioral Deficits in Animal Modelsof Parkinson's Disease

Materials and Methods

Chemical Library.

The 960 compounds from The Genesis Plus Collection (MicroSourceDiscovery Systems) were used to identify the Nurr1 activator. Thecompounds in this library are primarily U.S. Food and DrugAdministration-approved compounds. The 720 compounds containing purenatural products and their derivatives (Microsource Discovery Systems)were also tested.

Plasmid Constructs.

The pTH2600LUC reporter construct contains the 2.6 kb upstream sequencesof the rat tyrosine hydroxylase gene in front of a firefly luciferasegene in pGL3-basic vector. A Nurr1-expression plasmid, pSV40Nurr1, waskindly provided by Dr Orla M. Conneely at Baylor College of Medicine(Houston, Tex., USA). Full-length mouse Nurr1cDNA was amplified byRT-PCR from pSV40Nurr1 using forward5′-GCTTTCAAGCTTATGCCTTGTGTTCAGGCGCAGTATGG-3′ (SEQ ID NO: 1) and reverse5′-GTCAATCTCGAGGAAAGGTAAGGTGTCCAGGAAAAG-3′ (SEQ ID NO: 2)oligonucleotides. The amplified PCR fragment was cut with Hind III andXho I and then cloned into the plasmid pCDNA3.1/myc-His (Invitrogen),resulted in the expression of a C-terminal myc-tagged fusion protein andnamed with pCMVNurr1-myc. The Nurr1 LBD fragment from amino acidposition 328 to amino acid position 598 was PCR-amplified and subclonedinto pZeoSV (Invitrogen) containing the GAL4 DNA binding domain. TheNur77 LBD (aa 354-598), glucocorticoid receptor-a (GRα) LBD (aa522-777), liver X receptor-a (LXRa) LBD (aa 207-447), retinoid Xreceptor (RXR) LBD (aa 223-462), peroxisome proliferator-activatedreceptor-a (PPARα) LBD (aa 201-467), peroxisome proliferator-activatedreceptor-g (PPARγ) LBD (aa 209-476) were amplified by PCR and subclonedto pZeoSV GAL containing the GAL4 DNA binding domain. The reporterplasmid, p8xGAL-Luc, was constructed by cloning syntheticoligonucleotides to generate 8 tandem repeat GAL4 binding sites(5′-CTCGGAGGACAGTACTCCG-3′ (SEQ ID NO: 3))¹ upstream of the luciferasein pGV-B2 (Toyo Ink) plasmid. 4 copies of NL3(5′-GATCGAAAACAAAAGGTCACTTAC-3′ (SEQ ID NO: 4); bold indicates aconsensus NBRE motif)² were inserted into upstream of the minimalpromoter of TATA-Luciferase, resulting in p4x(NL3)-Luc. Mouse SRC-1 andhuman SRC-3 cDNAs were amplified by PCR using pYX-mSRC1 (Open Biosystem,MMM1013-9498102) and pCR-TOPO hSRC3 (Open Biosystem, MHS4426-99625636)as a template, respectively. Amplified cDNAs were subcloned into pCMV 2B(Stratagene) and resulted in the expression of an NH2-terminalflag-tagged fusion protein. The VP16/SRC1 and VP16/SRC3 hybrid wereconstructed by inserting the transactivation domain of the VP16 proteinupstream of SRC1 and SRC3, respectively. Nurr1-LBD, RXR-LBD, andNur77-LBD were amplified by PCR using 5′-AAAAAACATATGGTTAAAGAAGTGGTTCG-3′ (SEQ ID NO: 5) and5′-GTCAATCTCGAGTTAGAAAGGTAAGGTGTCCAGGAAAAG-3′ (SEQ ID NO: 6) for Nurr1LBD, 5′-GAG GTG CAT ATG ACC AGC AGC GCC AAC GAG GAC ATG-3′ (SEQ ID NO:7) and 5′-AAA AAA CTC GAG CTA AGT CAT TTG GTG CGG CGC CTC-3′ (SEQ ID NO:8) for RXR LBD, and 5′-AAA CCC CAT ATG AAG CAG CCC CCA GAT GCC-3′ (SEQID NO: 9) and 5′-AAA AAA CTC GAG TCA GAA GGG CAG CGT-3′ (SEQ ID NO: 10)for Nur77 LBD (bold letters indicate the restriction enzyme site) andthen subcloned into vector pET15b to express the His-tagged fusionprotein. The recombinant proteins were purified by Ni-NTA columnchromatography (Qiagen) according to the manufacturer's instruction. Theintegrity of all sequences was verified by DNA sequence analyses.

Transfection and Compound Screening.

Human neuroblastoma SK-N-BE(2)C cells were maintained in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.All culture media contained 100 units/ml penicillin and 100 μg/mlstreptomycin. For transfection, cells were plated at 1.5×10⁵ cells/wellin the DMEM media without antibiotic into 24-well plates 1 day prior totransfection. Transfections were carried out by the Lipofectamine plus(Invitrogen) according to the manufacture's protocol. Total DNA amountwas 0.5 μg per well with 0.1 μg of pRSV-β-gal as an internal control. 6hours after transfection, compounds in the DMEM media with 1%charcoal-stripped fetal calf serum was added and incubated overnight.Cells from each well were lysed with 100 μl of lysis buffer, whichcontains 25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM CDTA(1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid), 10% glycerol, and1% Triton X-100. Then equal volume of firefly luciferase substrate wasadded and the luciferase activity was measured using a Luminometer platereader and normalized for beta-galactosidase activity.

Western Blotting.

Cells were washed twice with phosphate-buffered saline (PBS) andharvested in a solution containing 1% Triton X-100, 20 mM Tris (pH7.6),150 mM sodium chloride, 1 mM phenylmethylsulfonylfluride (PMSF). Thecell suspension was sonicated and boiled in an equal volume ofSDS-sample buffer. Samples were subjected to SDS-polyacrylamide gelelectrophoresis (PAGE) and transferred to a nitrocellulose membrane(Hybond-ECL, Amersham). After blocking, the membrane was incubated withprimary antibodies diluted in PBS containing 0.1% BSA. The followingprimary antibodies were used: rabbit anti-Nurr1 (Santa Cruz; 1:3000),mouse anti-myc (Roche; 1:3000), mouse anti-flag (Roche 1:3000), rabbitanti-caspase3 (Cell Signalling; 1:1000), and mouse anti-actin (Sigma1:5000). The membrane was incubated with 1:3000-dilution of horseradishperoxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG)antibody (Amersham). Detection was achieved using anenhanced-chemiluminescent substrate (Amersham).

Immunoprecipitation Analysis.

Cells were disrupted in lysis buffer containing 20 mM Tris (pH7.6), 100mM sodium chloride, 0.5% NP-40, 0.5 mM EDTA, 0.5 mM PMSF, 1× proteaseinhibitor cocktail (Roche). One μg of anti-flag previously absorbed for4 hours with protein-Aagarose (Upstate) were added to media and celllysates, and incubated for 16 hours at 4° C. Pellets were collected bycentrifugation for 5 minutes and washed 3 times with lysis buffer. Theimmunoprecipitated proteins were released from protein-A by boiling 5minutes in SDS-sample buffer. Samples were analyzed by 10% SDS-PAGE andsubjected to western blotting assay using mouse anti-myc antibodies.

BIAcore Analysis.

Binding experiments were performed with the surface PlasmonResonance-based biosensor instrument BIAcore 3000 (BIAcore AB, Uppsala).Immobilization of the purified proteins on the sensor surface wasperformed following the standard amine coupling procedure according tomanufacturer's instructions. The final immobilization response of eachprotein was 6000 resonance units. A reference surface was generatedsimultaneously under the same conditions with BSA and used as a blank tocorrect for instrument and buffer artifacts. AQ and kanamycin wereinjected at variable concentrations at 20 μl/min flow rate, and bindingto the protein immobilized on the chip was monitored in real-time. Eachsensorgram consists of an association phase (60-160 sec), reflectingbinding of the injected AQ to the protein, followed by a dissociationphase (after 160 sec), during which the running buffer is passed overthe chip and the bound AQ is being washed off the protein surface.Binding data were analyzed using the BIAcore 3000 CONTROL® software andthe BIAEVALUATION® software.

Fluorescence Quenching Assay.

Fluorescence spectra were measured using a SHIMADZU fluorescencespectrophotometer (model RF-5310PC, SHIMADZU). The proteins (15 ng/μl)were incubated with different concentrations of AQ or kanamycin (0.1 nM,1 nM, 10 nM, 100 nM and 1 μM in PBS buffer, pH7.0). Protein quenchingwas monitored at 25° C. by using 5 nm of excitation and 5 nm of emissionslit-width. The excitation wavelength was 280 nm, and the emissionspectra were measured between 290 and 430 nm.

Filter Binding Assay.

Aliquots (100 μl) of an assay mixture containing 0.32 μM Nurr-LBD and3.9-1000 nM [³H]-CQ (Moravek Biochemicals, CA) in binding buffer (25 mMsodium acetate buffer (pH 5.2)) were incubated at 4° C. for overnight.Non-specific binding was determined in parallel in the presence of a1000-fold molar excess of unlabeled CQ. GF/C filters were wet with inbinding buffer and placed on a 48-place filter manifold. 100 μl of assaymixture containing the [³H]-CQ and Nurr1-LBD was applied on the surfaceof the filter. Filters were washed with 4×100 ml binding buffer, dried,and mixed with 5 ml Scintiverse BD (Fisher) and radioactivity wasdetermined in a Beckman LS6000SC liquid scintillation counter. Forcompetition assay, reactions containing 0.32 μM Nurr-LBD, 500 nM [³H]-CQand various concentrations of the competitor were incubated at 4° C. forovernight. The mixture was applied to GF/C filter and filter was washedand counted as described above.

Dopamine-Uptake Assay.

For measuring [³H]Dopamine uptake following 6-OHDA treatment in thepresence or absence of AQ, DA neurons were rinsed with Krebs Ringerssolution, incubated in Krebs Ringers solution containing 1 mM ascorbate,2 mM β-alanine, 100 μM pargyline at 37° C. for 5 min in a humidified 5%CO₂ atmosphere, and further incubated with 25 nM [³H]Dopamine (45Ci/mmol; Amersham Pharmacia) for 15 min. Control DA neurons were treatedwith the above medium in the presence of a dopamine uptake blocker, 10μM mazindol (Sigma). Cells were then washed with ice-cold Krebs Ringerssolution and lysed with 0.5 M NaOH. Radioactivity was quantified by aliquid scintillation counter (Beckman, Fullerton, Calif.).

Cell Culture and Drug Treatments.

PC12 cells were initially incubated at a density of 2×10⁵ cells inpoly-D-lysine-coated 24-well dishes and subsequently switched toserum-free N-2-defined medium containing 6-OHDA with or without AQ. Cellviabilities were determined by the conventional MTT reduction assay.After incubation for indicated times, cells were treated with the MTTsolution (final concentration, 1 mg/ml, Sigma) for 1 h. Formazan grainsformed in intact cells were solubilized with MTT Solubilization.Solution containing 10% Triton X-100 plus 0.1 N HCl in anhydrousisopropanol for 24 h and then absorbance at 570 nm was measured with amicroplate reader (Molecular Devices, Menlo Park, Calif.).

To prepare primary cultures of DA neurons, the ventral mesencephalon wasremoved from 14 day gestation Sprague Dawley rat embryo (Charles River,Cambridge, Mass.). Tissues were incubated with 0.01% trypsin in MEM(MEM, Invitrogen) for 10 min at 37° C. and triturated using aconstricted Pasteur pipette. DA neurons were plated at 1.0×10⁵ cells per9 mm diameter glass cover slip precoated with 100 μg/ml poly-D-lysine(Sigma, St. Louis, Mo.) and 4 μg/ml laminin (Invitrogen). DA neuronswere maintained at 37° C. in a humidified 5% CO₂ atmosphere in minimumessential medium supplemented with 10% FBS (Hyclone), 2 mM glutamine and6.0 g/l glucose. At 5 or 6 days in vitro, DA neurons were washed withMEM and treated with various experimental reagents for the time periodsindicated. Reagents used were 6-OHDA (Sigma), N-acetyl-cysteine,N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fink; EnzymeSystems Products, Livermore, Calif.). BV-2 cells, a microglial-likemouse cell line, were thawed and passaged until logarithmic growth wasachieved. Cell cultures were maintained in 6-well culture plate (CorningCostar, Corning, N.Y.) at a concentration of 1.0×10⁵ cells per well, inthe growth medium consisting of DMEM (Sigma, St. Louis, Mo.),supplemented with 10% fetal bovine serum (FBS; Hyclone) heat inactivatedat 56° C. for 30 min, 2 mM L-glutamine(Sigma) and 1× Pen-Strep(Invitrogen). To begin the experiment, BV-2 cells were washed with DMEMand treated with LPS (Sigma) in the presence or absence of AQ (Sigma).The plates were at 37° C. in an atmosphere of 5% CO₂/95% air for thelisted period of time. Cells from each single well were withdrawn forRNA extraction.

Neural precursor cells (NPCs) from dissection of rat embryos days 14cortices (E14) were proliferated in N2 medium supplemented with 20 ng/mLbasic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, Minn.,USA). Following 3-4 days of bFGF-expansion, NPCs were dissociated inCa²⁺/Mg²⁺-free Hank's balanced salt solution (CMF-HBSS, Invitrogen,Carlsbad, Calif.) and re-plated onto coverslips (12 mm diameter;Carolina Biological Supply Company, Burlington, N.C.) at 32,000cells/coverslip pre-coated with poly-L-ornithine (PLO; 15 mg/ml, Sigma,St Louis, Mo.) at 37° C. overnight followed by fibronectin (FN; 1 mg/ml,Sigma) overnight. Prior to drug treatment, NPCs were transduced withNurr1-expressing retrovirus of MOI 5 in the presence of polybrene (1μg/ml, Sigma), cultured overnight in N2, and then differentiated.Getting started chemical treatment (every day for 2 hours), bFGF was notadded anymore³.

Immunocytochemistry and Immunohistochemistry.

Cells were fixed in 4% paraformaldehyde (PFA) for 20 min, washed andthen incubated with blocking buffer [PBS, 10% goat serum (Invitrogen),0.3% Triton X-100 (Sigma)] for 1 hour. Cells were then incubatedovernight at 4° C. with primary antibodies with the primary antibody inPBS containing 1% normal goat serum and 0.1% Triton X-100. A rabbitpolyclonal anti-TH antibody (1:500; Pel-Freez, Rogers, Ark.) was used asa primary antibody. Following washes with PBS, appropriatefluorescence-tagged secondary antibodies (Jackson ImmunoresearchLaboratories, West Grove, Pa.) were used for visualization. Stainedsamples were mounted in VECTASHIELD with DAPI mounting solution (VectorLaboratories INC., Burlingame, Calif.) and photographed usingepifluorescence microscope (Leica). The animals were euthanized byanesthesia with pentobarbital (3 ml/kg), and then transcardiallyperfused with 4% PFA in a 0.05M phosphate buffer (PB).Immunohistochemistry and immunofluorescence assay were performed aspreviously described⁴, on free-floating cryomicrotome-cut sections (40μm thick) that encompassed the entire brain. After their incubation with3% H₂O₂ in 0.05M PBS, followed by 0.3% Triton X-100 and 3% bovine serumalbumin (BSA) in 0.05M PBS, the sections were stained overnight at 4° C.with a primary antibody. The ABC kit (Vector Laboratories., Burlingame,Calif., USA) was used as a secondary antibody. After the tissue waswashed three times in 0.05M PBS, followed by 0.3% Triton X-100 and 1%BSA in 0.05M PBS, the sections were stained overnight at 4° C. withprimary antibodies. The following primary antibodies were used: mouseanti-Iba-1 (Abcam; 1:500), goat anti-FoxA2 (SCBT; 1:200), sheepanti-tyrosine hydroxylase (TH) (Pel-Freez; 1:200), rabbit anti-TH(Pel-Freez 1:200), goat anti-FoxA2 (Millipore; 1:200), and sheepanti-AADC (Millipore; 1:500). The tissues were washed three times in PBSand incubated for 2 h with the secondary fluorescence-conjugated goatanti-mouse and rabbit antibody (DAKO, Denmark, Cy3, FITC, both dilutionsat 1:1,000) and tissues were washed and mounted in Vectorshield. Alltissue samples were additionally counterstained with DAPI. Tissues werevisualized using fluorescence microscopy. Every 6^(th) section of ratbrains was assayed to count immunoreactive cells using the Image J(version 1.42q, National Institutes of Health, USA) and StereoInvestigator and software (Microbright Field, Williston, Vt.). Theprotection percentage of total TH⁺ cells in the lesioned hemisphere ofeach rat brain was calculated by dividing total TH⁺ cells in the intacthemisphere of each rat brain and multiplying 100⁵.

Semi-Quantitative and Quantitative RT-PCR Analyses.

Total RNA was extracted by single step preparation using Trizol reagent(Invitrogen) following the manufacturer's instructions. Total RNA fromeach single well was reverse transcribed into cDNA using SuperscriptIIReverse Transcriptase and oligo(dT)18 primer (SEQ ID NO: 35)(Invitrogen). Real-time PCR analysis was performed using theABI-Prism7700 sequence detection system (Applied Biosystems). Briefly,it was performed in a 0.5 ml tubes (Applied Biosystems) on cDNAequivalent to 50 ng of DNase-digested RNA in a volume of 25 μl,containing 12.5 μl of SyBR green Universal Master mix, forward andreverse primers (IL-1β; forward 5′-GCA ACT GTT CCT GAA CTC AAC T-3′ (SEQID NO: 11), reverse 5′-ATC TTT TGG GGT CCG TCA ACT-3′ (SEQ ID NO: 12),IL-6; forward 5′-TAG TCC TTC CTA CCC CAA TTT CC-3′ (SEQ ID NO: 13),reverse 5′-TTG GTC CTT AGC CAC TCC TTC-3′ (SEQ ID NO: 14) iNOS; forward5′-ACA TCG ACC CGT CCA CAG TAT-3′ (SEQ ID NO: 15), reverse 5′-CAG AGGGGT AGG CTT GTC TC-3′ (SEQ ID NO: 16), TNF-α; forward 5′-ATG TCG GCT CCAGGA CCT TA-3′ (SEQ ID NO: 17), reverse 5′-GGT AGT AAC TGT TGA CAC CCACT-3′ (SEQ ID NO: 18), mouse GAPDH; forward 5′-AGG TCG GTG TGA ACG GATTTG-3′ (SEQ ID NO: 19), reverse 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′(SEQ ID NO: (20), TH; forward 5′-AGC CCC CAC CTG GAGTAT TTT G-3′ (SEQ IDNO: 21), reverse 5′-AGC AAT CTC TTC CGC TGT GTA TTC-3′ 22), AADC;forward 5′-GCC TTT ATC TGT CCT GAG TTC CG-3′ (SEQ ID NO: 23), reverse5′-TGA TGA GTC CTG AGT CCT GGT GAC-3′ (SEQ ID NO: 24), DAT; forward5′-GCT GGC ACA TCT ATC CTC TTT GG-3′ (SEQ ID NO: 25), reverse 5′-CAA TGCTGA CCA CGA CCA CAT AC-3′ (SEQ ID NO: 26), VMAT; forward 5′-GCA CAC AAAATG GGA AGG TGG C-3′ (SEQ ID NO: 27), reverse 5′-CAT TTT TTC CTC CTT AGCAGG TGG-3′ (SEQ ID NO: 28), rat GAPDH; forward 5′-TGA CAT CAA GAA GGTGGT GAA GC-3′ (SEQ ID NO: 29), reverse 5′-GGA AGA ATG GGA GTT GCT GTTG-3′ (SEQ ID NO: 30)) following manufacturer's protocol. Gene expressionvalues were normalized to those of GAPDH.

Chromatin Immunoprecipitation

PC12 cells were grown in 150 mm dishes in the absence or presence of 20μM AQ or 75 μM CQ for 15 hrs. Cells were washed with PBS, fixed with 1%formaldehyde, lysed, and sonicated according to the manufacturer'sinstruction (Active Motif). 25 μg of soluble chromatin DNA wereco-immunoprecipitated 1 μg of Nurr1 specific antibody (E-20, Santa Cruz)or control antibody (rabbit IgG, santa Cruz). 2 μl ChIP DNA wassubjected to quantitative real-time PCR using the following primerpairs: NL1: 5′-GCG TGG AGA GGA TGC GCA GG-3′ (SEQ ID NO: 31) and 5′-AGTGCA AGC TGG TGG TCC CG-3′ (SEQ ID NO: 32); NL3: 5′-TCC TTA GAG ATC CTGTTT CC-3′ (SEQ ID NO: 33) and 5′-TCA GCT GGT CCC CAT GTA AG-3′ (SEQ IDNO: 34). Values are shown as fold induction compared to mock treatedcontrol cells.

6-OHDA-Lesioned Rat Model of PD.

To assess the potential neuro-protective effects of AQ/CQ, we generatedintrastriatal 6-OHDA-lesioned rats⁶⁻⁸ using male Sprague-Dawley ratsweighing 240-300 g at the beginning of experiments. The rats wereanesthetized with Ketamine [4.5 mg/kg, intraperitoneal (i.p.)] mixedwith Rumpun (1.5 mg/kg, i.p.) and given a unilateral stereotaxicinjection of 6-OHDA (20 μg/5 μl with 0.2 mg/ml L-ascorbic acid; Sigma,St. Louis, Mo., USA) or the same volume of the vehicle (L-ascorbic acid)into their right striatum using coordinates relative to the bregma anddura (A/P, 0.7; L/M, −2.6; D/V, −4.5) according to the atlas of Paximosand Watson⁹ at the rate of 1 μl/min using a 26-gauge Hamiltonmicrosyringe. Following infusion, the cannula was kept in place for 5min to minimize backflow before being slowly retracted. The use ofanimals was in accordance with McLean's Institutional Animal Care andUse Committee and followed National Institutes of Health guide lines. Inour preliminary experiments, we tested different doses (1, 5, and 20mg/kg) of both AQ and CQ and found that AQ at 20 mg/kg exhibited mostprominent effects. Thus, we used this condition for more systematic invivo experiments.

Assay for the Neuroprotective Effects of AQ in the 6-OHDA-Lesioned RatModel.

One day before modeling PD, rats were randomly assigned to two groups:saline group (n=6) and AQ-treatment group (n=8). AQ-treatment groupreceived an i.p. injection of 20 mg/kg of AQ twice one day before 6-OHDAinjection. After 6-OHDA injection, animals were given either AQ orsaline twice per day for 13 days (in total, 2 weeks of AQ injectionincluding one day of AQ injection before the PD modeling). For thebehavioral functional assay, rotation behavior was measured for 90 minfollowing intraperitoneal injection of 5 mg/kg amphetamine(Sigma-Aldrich, St. Louis, Mo.), using automatized rotameter system (MedAssociate Inc., St. Albans, Vt., USA)⁶′⁷. Ipsilateral rotations weremeasured at 4 and 6 weeks post 6-OHDA lesioning.

Abnormal Involuntary Movements (AIMs) Behavior in 6-OHDA-Lesioned Rats.

To test dyskinesia-like involuntary behaviors, AIMs test was performedat the two time points, immediately after two weeks of drug injection (2weeks post 6-OHDA lesion; n=6), and one month after two weeks of druginjection (6 weeks post 6-OHDA lesion; n=6). Saline- and L-DOPA (8 mg/kgfor two weeks; n=6) treatment was used as negative and positive controlof AIMs behavior, respectively. The following behavioral tests were allvideotaped and scored at a later date by two investigators blind to theanimals' treatment. Rats were monitored for four types of AIMs¹⁰; Axial(dystonic posturing of neck and torso twisted toward the side of thebody contralateral to the lesion), Forelimb (contralateral forelimbmovements that are “rapid” and “purposeless”), Orolingual (repetitivejaw openings and tongue protrusions), and Locomoter (walking in acontralateral circular motion) AIMs. Rats were observed for 2 minutes atevery 20 minute mark within a 2 hour period. During the 1^(st) minute,each AIMs behavior was monitored and scored with 0-4 scale (0: noabnormal behavior at all; 1: present for less than 50% of observationtime; 2: present for over 50% of observation time; 3: present for theentire observation period but interrupted by external stimulus; 4:present for the entire observation period and not interrupted byexternal stimulus). The AIMs scores for the 2 hour period in eachcategory were summed (maximum score is 24).

Behavioral Studies in Ak/Ak Mice.

Either 20 mg/kg of AQ (AQ group) or the saline (Saline group) wasintraperitoneally injected (I.P.) twice per day for two weeks. 8 mg/kgof L-DOPA (L-DOPA group) in 0.5% DMSO was injected (I.P.) once per dayfor two weeks. The L-DOPA group was given 12.5 mg/kg benserazide 20 minprior to administration of L-DOPA.

Behavioral Experimental Groups and Behavioral Testing Design:

Each ak mouse was randomly assigned into one of three experiment groups,(1) saline group (negative control; n=8), (2) L-DOPA group (positivecontrol; n=8), (3) AQ group (n=10). Each WT or rdl mouse was randomlydivided into two groups, (1) saline group (n=8) and (2) AQ group (n=10).Pole Test, Challenging Beam Traversal Test, and Cylinder Test wereperformed at two time points, immediately and one month after two weeksof either AQ or saline injection. Each mouse ran each task twice. Rotarod test was performed immediately after two weeks of either drug orsaline injection. Acclimation and data recording process were identicalto the rat study described above. All tasks were performed as describedpreviously¹¹ and the short description of each task is as follows.

Pole Test:

A vertical wooden pole 50 cm in length and 1 cm in diameter was used forPole Test. Each mouse was gently placed head upwards on top of the polewhich is placed on each animal's home cage. Each mouse ran tree trialsper session during 2 training sessions. On the testing day, mice ranthree trials. Two parameters (the meantime to orient downward and thetotal travel mean time) were used for the statistical analysis.

Challenging Beam Traversal Test:

A 1 m length of plastic beam was used for Challenging Beam TraversalTest. The starting width of the beam was 3.5 cm and it graduallynarrowed in 1 c increments to 0.5 cm at the other end site of the beam.Animals were trained to walk from the wide end to the other end for 2sessions, three trials per session before testing. A mesh grid wasremoved during the training period. On the testing day, a mesh grid (1cm square) covering the beam surface was equipped leaving a −1 cm roombetween the grid and the beam surface. On the testing day, mice ranthree trials. Two parameters (the mean number of steps taken by eachanimal and the mean time to traverse across three trials) were collectedand used for statistical analysis.

Cylinder Test:

A small transparent cylinder (height, 15.5 cm; diameter, 12.7 cm) wasused to evaluate somatosensory motor function. Mice were placed in thecylinder and their rears were counted for 5 minutes. The forelimbtouching the Plexiglas wall (a vertical movement) is defined as a fullrear. The mean number of forelimb use was calculated for the statisticalanalysis. Each mouse ran Cylinder task for one session and no trainingsessions were necessary.

Rota Rod Test:

The Rota rod equipment (Med-Associates, Inc, Albans, Vt., USA) was usedto measure motor coordination. The Rota rod is accelerated from 4 to 40revolutions per minute (RPMs) over 4 min. The mean latency to fall offfrom the Rota rod was measured for the statistical analysis. Each mouseruns 3 consecutive trials daily with 10 min interval between trials for3 consecutive sessions.

Statistical Methods

Statistical analyses were conducted using the statistical AnalysisSystem (Version 9.1; SAS institute, Cary, N.C.). Performance measures ofin vitro outcomes, cell counting, and behavioral outcomes were analyzedusing the PROC TTEST, ANOVA, MIXED or GLIMMIX program, a generalizedlinear mixed models procedure for conducting repeated measures analysesfollowed by LSD (Fisher's least significant difference) post-hoc tests(LSD is a default post-hoc test provided in SAS). Means were calculatedfor each animal for each testing condition, defined by the followingvariables (where appropriate): Treatment (AQ, L-Dopa, or saline) andTime (2, 4, or 6 weeks after 6-OHDA lesion).

REFERENCES

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RESULTS AND DISCUSSION

Based on its unique dual role for development/maintenance of midbraindopamine neurons and for their protection from inflammation-induceddeath, Nurr1 is a potential target for Parkinson's disease (PD). Here weshow successful identification of Nurr1 agonists that share an identicalchemical scaffold and stimulate the transcriptional activity throughbinding to the ligand-binding domain. Remarkably, they also enhanced thecontrasting dual functions of Nurr1 and improved behavioral deficits inanimal models of PD.

PD, primarily caused by selective degeneration of midbrain dopamine(mDA) neurons, is the most prevalent movement disorder, affecting 1-2%of the global population over the age of 65¹⁻³ Currently availablepharmacological treatments (e.g., L-DOPA) are largely symptomatic andlose their efficacy over time, with accompanying severe side effectssuch as dyskinesia. Thus, there is an unmet clinical need to developmechanism-based and/or disease-modifying treatments^(2,3). The orphannuclear receptor Nurr1 (also known as NR4A2) is essential not only fordevelopment and maintenance of mDA neurons⁴⁻⁷ but also for theirprotection from inflammation-induced death⁸. Furthermore, previousstudies showed decreased Nurr1 expression in postmortem brains of PDpatients⁹ and functional mutant forms in rare familial PD cases¹⁰,strongly suggesting that Nurr1 is a promising target for the developmentof novel disease-modifying therapeutics for PD″. To address thispossibility, we established efficient high-throughput assay systemsbased on Nurr1's ability to directly activate TH promoter function¹².Using these cell-based assay systems, we screened a library composed of960 FDA-approved drugs and successfully identified three hit compounds,i.e., two antimalarial drugs amodiaquine (AQ) and chloroquine (CQ) andthe pain drug glafenine. Surprisingly, all three compounds share anidentical chemical scaffold, 4-amino-7-chloroquinoline, suggesting astructure-activity relationship (SAR) (FIG. 13a ).

In inventors' previously reported assays using the reporter constructp4xNL3-Luc, containing four copies of the NBRE-like NL3 motif residingin the TH promoter¹², AQ and CQ activated luciferase reporter activityup to approximately 3-fold in a dose-dependent manner (FIGS. 3B, 15A,and 16B). In the present study the inventors focused on AQ and CQ sinceglafenine showed a weaker activity (approximately 1.6-fold). Toinvestigate whether AQ and CQ activate Nurr1 function through its ligandbinding domain (LBD) or DNA binding domain (DBD), the inventors nextgenerated additional reporter constructs in which the yeasttranscription factor GAL4's DBD is fused to either Nurr1 LBD or DBD(pSVGAL_((DBD))-N_((LBD)) and pSVGAL_((DBD))-N_((DBD))). Notably, AQ andCQ stimulated Nurr1's transcriptional activity through its LBD (FIG.3B), while no response was observed when GAL4 or GAL4-Nurr1 DBD wasused. AQ and CQ induced Nurr1 LBD-based reporter activity up-to 15- and10-fold with an EC₅₀ of approximately 20 and 50 μM, respectively (FIGS.3B, 16C, and 16D).

To test if AQ and CQ act through Nurr1, the inventors treated cells withNurr1-specific siRNA. Nurr1 siRNA, but not scrambled RNAs, reducedluciferase activation by >60%, suggesting that transcriptionalactivation by AQ and CQ is indeed through modulation of Nurr1 function(FIGS. 3C and 17). The inventors next tested if various quinolinecompounds have similar Nurr1-activating function. Remarkably, none ofthese compounds showed any detectable transactivation function in a widerange of concentrations (FIG. 18). Since only AQ and CQ contain the4-amino-7-chloroquinoline entity, these results further support its SAR.In addition, AQ and CQ were unable to induce the transcriptionalactivity of other nuclear receptors (NRs), showing high selectivity(FIG. 19).

A crucial mechanism of NRs is the recruitment of transcriptionalcoregulators as seen with steroid receptor coactivators (SRCs)¹³. Totest if AQ and CQ modulate Nurr1's interaction with SRC, aco-immunoprecipitation assay was performed. We found that Nurr1's weakinteractions with SRC-1 and SRC-3 were significantly enhanced by AQ andCQ (FIG. 13D). We next tested the effects of AQ and CQ on Nurr1transcriptional function following SRC-1 or SRC-3 overexpression. WhileSRC-1/SRC-3 overexpression itself did not greatly influence Nurr1function, AQ and CQ further enhanced Nurr1's transcriptional function inthe presence of SRC-1/SRC-3 overexpression (FIG. 13E), suggesting thatAQ and CQ induce Nurr1's transactivation function through its Nurr1 LBDby facilitating the recruitment of coactivators like SRC-1/SRC-3.

To investigate whether AQ and CQ physically interact with Nurr1's LBD,the inventors purified the Nurr1 LBD polypeptide (amino acid 328-598)and analyzed their physical binding using the Biacore S51 SPR sensor,which has a higher sensitivity and improved fluidics, enabling themonitoring of small signal changes derived from binding of compounds toproteins.

As shown in FIGS. 13F and 20A, AQ specifically bound to Nurr1-LBD in adose-dependent manner but not to the LBDs of other NRs such as Nur77 orRXR. Next, we performed fluorescence quenching analysis. The Nurr1-LBDdisplayed maximal fluorescence at 336 nm whereas AQ itself had nofluorescence at this wavelength. When the Nurr1-LBD was incubated withincreasing amounts of AQ, fluorescence intensity gradually decreased(FIG. 3G). In contrast, Nur77-LBD and RXR-LBD's fluorescence emissionwas not quenched by AQ over a wide range of concentrations (FIGS. 3G and20B). In addition, the inventors performed a radioligand binding assayusing [³H]-CQ. The [³H]-CQ showed saturable binding to Nurr1-LBD with adissociation constant (K_(a)) of 0.27 μM and a maximal binding capacity(B.) of 13.9 μM (FIG. 13H). Furthermore, competition-binding assayshowed that unlabeled AQ/CQ can compete for binding with [³H]-CQ with K,values of 246 and 88 nM for AQ and CQ, respectively (FIG. 13I). Asexpected, unlabeled primaquine which couldn't enhance the Nurr1transcriptional function (FIG. 18) was unable to compete. Together,these data strongly suggest that AQ/CQ activate Nurr1 function viadirect and specific binding to Nurr1's LBD.

We next sought to test whether these compounds exhibit biologicaleffects on mDA neurons in more physiological contexts. First, we testedif they can increase the generation of TH⁺ neurons and/or expression ofmDA-specific genes during in vitro differentiation of neural stem cells.We found that AQ enhanced Nurr1's function both in terms of the numberof TH⁺ neurons generated from neural stem cells and mRNA expressionlevels of the TH, dopamine transporter (DAT), vesicular monoaminetransporter (VMAT), and aromatic amino acid decarboxylase (AADC) genes(FIGS. 14A, 14B, and 21). In vivo chromatin immunoprecipitation (ChIP)assay clearly showed that Nurr1 was recruited to both NL1 and NL3 sitesof the TH promoter in AQ- and CQ-treated cells, suggesting that Nurr1 isdirectly involved in AQ- and CQ-mediated induction of TH expression(FIG. 14C). Second, we found that AQ and CQ significantly inhibitneurotoxin (6-OHDA)-induced death in primary DA neurons, as examined bythe number of TH⁺ neurons (FIG. 14D) and DA uptake (FIG. 14E). Theneuroprotective effect of AQ and CQ was also observed in rat PC12 cells(FIG. 14F). Since Nurr1 has opposite transrepression activity inmicroglia and astrocytes⁸, the inventors also analyzed the effect of AQon expression of pro-inflammatory cytokine genes in primary microgliaderived from P1 rat brains. When these cells were treated with theinflammation-inducing lipopolysaccharide (LPS; 10 ng/ml) for 8 hrs,expression of all cytokine genes tested (IL-1β, IL-6, TNF-α and iNOS)was dramatically induced (FIG. 14G). Remarkably, AQ treatmentprominently reduced the expression of all these genes in a dosedependent manner (>10-fold). AQ and CQ showed very similar effects forrepressing these cytokine genes in both primary microglia and BV-2microglial cell line (FIG. 22). Taken together, our data show that AQand CQ are able to enhance the contrasting dual roles of Nurr1: (1) theyincrease Nurr1's transactivation of mDA-specific genes in mDA neuronsand (2) they also further enhance Nurr1's transrepression ofpro-inflammatory cytokine gene expression in microglia.

The above findings prompted the inventors to test whether thesecompounds can ameliorate motor behavior deficits by neuroprotectivemechanism(s) in PD animal models. Toward this goal, we tested theeffects of AQ administration in an animal model of PD, 6-OHDA lesionedrats. Rats with unilateral intrastriatal 6-OHDA lesion were administeredwith saline or AQ for two weeks, starting from 1 day prior tointrastriatal 6-OHDA lesion (FIG. 15A). As expected, amphetamineadministration produced rotation behavior toward the lesion side incontrol rats, when measured at 4 and 6 weeks post 6-OHDA lesion,indicating unilateral damage to the right striatum (FIGS. 15A and 15B).Remarkably, AQ administration significantly ameliorated 6-OHDA-inducedrotation behavior at 4 and 6 weeks post 6-OHDA lesion (p<0.06 andp<0.0003, respectively). Immunohistochemistry of the substantia nigra(SN) and the striatum (STR) of these animals showed that AQ-treated ratsretained a significant number of TH⁺ cells in the lesion side SN whilesaline-treated control rats lost the great majority of TH⁺ cells (FIG.15C). Similarly, abundant TH⁺ fibers were spared in the STR ofAQ-treated brains while they were mostly lost in the STR of controlbrains. To quantitatively analyze these data, TH⁺ cells in the SN werestereologically counted in a blind manner. As shown in FIG. 15D, thenumber of TH⁺ neurons in the lesion side of AQ-treated rats wasapproximately 60% of those from the intact side at 6 weeks post 6-OHDAlesion (p<0.0003), whereas it remained less than 20% in saline-treatedanimals. Importantly, these spared TH⁺ cells co-expressed other DAmarkers such as FoxA2 and AADC (FIG. 23). The inventors also examinedmicroglial activation by immunohistochemistry of the microglial markerIba-1. As shown in FIGS. 15E and 24, microglial activation wasprominently observed in the ipsilateral SN and STR regions of 6-OHDAlesioned rats. In contrast, following AQ treatment, the numbers ofIba-1⁺ microglia in the ipsilateral SN and STR regions decreased to thelevels of the contralateral sides, indicating a robust suppression ofneuroinflammation by Nurr1 activation⁸. Without wishing to be bound by atheory, it is believed that Nurr1-based neuroprotective mechanism,unlike the L-dopa treatment, may not trigger dyskinesia-like sideeffects. To address this, the inventors tested if AQ treatment triggersabnormal involuntary movements (AIMs), well validated dyskinesia-likebehaviors¹⁴. Toward this end, the inventors measured AIM scores such asaxial, limb, locomotive, and orolingual dyskinesias. In L-DOPA-treatedrats (8 mg/kg for two weeks prior to scoring; FIG. 15A). The animalsshowed dramatically increased AIMs scores (F_((2,71))=33, p<0.0001),classified as the axial, limb, locomotive, and orolingual dyskinesias(FIG. 15F). In contrast, both saline- and AQ-treated 6-OHDA rats did notshow any detectable AIMs behavior.

The inventors next tested if AQ treatment can also improve motordysfunction in the aphakia mouse (ak/ak), that was shown to be a validmouse model of PD^(22,23). Since the great majority of A9 dopamineneurons (>80%) are selectively degenerated in the SN of the ak/ak mousestarting from birth, it can be considered to be a severe PD situation.Following 2 weeks of AQ treatment, the inventors performed a battery ofbehavioral tests sensitive to nigrostriatal impairment²². Remarkably,the inventors discovered that ak/ak mice's motor deficits weresignificantly improved in all these tests (i.e., Pole, Challenging Beam,Cylinder, and Rotarod tests) by AQ treatment (FIGS. 25A-25D). Notably,there was no group difference between AQ- and L-DOPA treated animalsindicating that AQ had similar effects as L-DOPA treatment on functionalrecovery in most tests, except the Cylinder test, in which L-DOPAtreatment dramatically induced rearing activity, as previouslyreported²². The number of TH⁺ neurons was not detectably altered in theAQ-treated ak/ak mice (data not shown). In contrast, the inventorsobserved robust microglial activation as examined by activatedmicroglial marker CD11b in the SN, which was reduced to the level ofwild type mice (FIG. 26). Thus, albeit no increased TH⁺ neurons in theSN, suppression of activated microglia appears to improve motor deficitsin ak/ak mice.

In summary, using the novel high throughput cell-based assay describedherein, the inventors identified two small molecules, AQ and CQ, whichcan activate Nurr1 through direct interaction with its LBD. Furthermore,AQ administration significantly improved motor impairments in 6-OHDAlesioned rodent models of PD without dyskinesia-like side effects. Thedata presented herein shows that these compounds enhanced thecontrasting dual functions of Nurr1—activation of mDA neuron-specificfunction (e.g., TH expression) and repression of microglial activationand neurotoxic cytokine gene expression—leading to a significantneuroprotective effect in a rodent model of PD. These findings aresurprising because Nurr1 lacks a ‘classical’ binding pocket due to thepresence of bulky hydrophobic side chain residues, and is thought to bea ligand-independent and constitutively active NR^(15,16). However,certain ligands are able to “worm” their way into the interior ofproteins and to squeeze into buried LBD by inducing or exploitingtransient conformational changes, thus creating a cavity^(17,18)suggesting the possibility that Nurr1 may become an “adopted” NR¹⁹.Notably, based on selective binding to neuromelanin of an MPTPmetabolite²⁰, a previous study demonstrated that administration of CQ tomonkeys protected them from MPTP-induced parkinsonian motorabnormalities²¹. Our findings are in line with this study showing CQ'sneuroprotective effect in a rodent or monkey model, and further suggestthe involvement of additional and/or alternate mechanisms related toNurr1 activation. Taken together, our results provide a preclinical“proof-of-concept” that Nurr1 could serve as a valid target for furtherdevelopment of mechanism-based, disease-modifying therapeutics of PD.

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All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

What is claimed is:
 1. A method of ameliorating symptoms of Parkinson'sdisease, the method comprising administering to a subject in needthereof a therapeutically effective amount of a compound selected fromthe group consisting of Formula (1)-Formula (X) and any pharmaceuticallyacceptable salt, hydrate, solvate, ester, stereoisomer mixture, orenantiomer thereof, where

wherein: X, Y, and Z are independently CR¹⁸, N, O, or S; Z¹ is N and Z²is CR¹⁴; R¹⁶ is chlorine; R¹⁰-R¹⁵ and R¹⁷-R¹⁸ are independently H,linear or branched alkyl, linear or branched alkenyl, linear or branchedalkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halogen,trifluoromethyl, alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy,amino, alkylamino, thiol, or alkylthio, each of which can be optionallysubstituted; and L is a linker

wherein: X and Y are independently CR¹⁸, N, O, or S; A and B areindependently CR¹⁸, N, O, or S; Z¹ is N and Z² is CR¹⁴; R¹⁶ is chlorine;R¹¹-R¹⁵ and R¹⁷-R¹⁸ are independently H, linear or branched alkyl,linear or branched alkenyl, linear or branched alkynyl, cyclyl,heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl, alkoxy, nitro,cyano, carbonyl, hydroxyl, phenoxy, amino, alkylamino, thiol, oralkylthio, each of which can be optionally substituted; ----- forms acyclyl, heterocyclyl, aryl, or heteroaryl, each of which can beoptionally substituted; and L is a linker;

wherein: A and B are independently CR¹⁸ or N; Z¹ is N and Z² is CR¹⁴;R¹⁶ is chlorine; R, R¹⁰-R¹⁵ and R¹⁷-R¹⁸ are independently H, linear orbranched alkyl, linear or branched alkenyl, linear or branched alkynyl,cyclyl, heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino, alkylamino,thiol, or alkylthio, each of which can be optionally substituted; and Lis a linker;

wherein: X and Z are independently CR¹⁸, N, O, or S; Z¹ is N and Z² isCR¹⁴; R¹⁶ is chlorine; R¹¹-R¹⁵ and R¹⁷-R¹⁸ are independently H, linearor branched alkyl, linear or branched alkenyl, linear or branchedalkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halogen,trifluoromethyl, alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy,amino, alkylamino, thiol, or alkylthio, each of which can be optionallysubstituted; ----- forms a cyclyl, heterocyclyl, aryl, or heteroaryl,each of which can be optionally substituted; and L is a linker;

wherein: X, Y, and Z are independently CR¹⁸, N, O, or S; Z¹ is N and Z²is CR¹⁴; R¹⁶ is chlorine; and R¹¹-R¹⁵ and R¹⁷-R¹⁸ are independently H,linear or branched alkyl, linear or branched alkenyl, linear or branchedalkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halogen,trifluoromethyl, alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy,amino, alkylamino, thiol, or alkylthio, each of which can be optionallysubstituted; and R¹⁰ is linear or branched alkyl, linear or branchedalkenyl, linear or branched alkynyl, cyclyl, heterocyclyl, aryl,heteroaryl, halogen, trifluoromethyl, alkoxy, nitro, cyano, carbonyl,hydroxyl, phenoxy, amino, alkylamino, thiol, or alkylthio, each of whichcan be optionally substituted;

wherein: X, Y, and Z are independently CR¹⁸, N, O, or S; R²⁷ ischlorine; R¹⁰, R¹¹, R¹⁸, R²¹-R²⁶ and R²⁸ are independently H, linear orbranched alkyl, linear or branched alkenyl, linear or branched alkynyl,cyclyl, heterocyclyl, aryl, heteroaryl, halogen, trifluoromethyl,alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy, amino, alkylamino,thiol, or alkylthio, each of which can be optionally substituted; and Lis a linker;

wherein: X, Y, and Z are independently CR¹⁸, N, O, or S; Z¹ is N and Z²is CR¹⁴; R¹⁶ is chlorine; and R¹⁰-R¹⁵ and R¹⁷-R²² are independently H,linear or branched alkyl, linear or branched alkenyl, linear or branchedalkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halogen,trifluoromethyl, alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy,amino, alkylamino, thiol, or alkylthio, each of which can be optionallysubstituted;

wherein: X, Y, and Z are independently CR¹⁸, N, O, or S; Z¹ is N and Z²is CR¹⁴; R¹⁶ is chlorine; and R¹⁰-R¹⁵ and R¹⁷-R²¹ are independently H,linear or branched alkyl, linear or branched alkenyl, linear or branchedalkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halogen,trifluoromethyl, alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy,amino, alkylamino, thiol, or alkylthio, each of which can be optionallysubstituted;

wherein: X, Y, and Z are independently CR¹⁸, N, O, or S; Z¹ is N and Z²is CR¹⁴; R¹⁶ is chlorine; and R¹⁰-R¹⁵ and R¹⁷-R²² are independently H,linear or branched alkyl, linear or branched alkenyl, linear or branchedalkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halogen,trifluoromethyl, alkoxy, nitro, cyano, carbonyl, hydroxyl, phenoxy,amino, alkylamino, thiol, or alkylthio, each of which can be optionallysubstituted;

wherein: R is hydrogen, CH₃, Cl, CF₃ or CN; R₁ is cyclic or acylicamine; and L is an optionally substituted C₂-C₃₀ alkyl, optionallysubstituted aromatic ring, optionally substituted heteroaryl ring,saturated cyclic ring; amino acid, or peptide.
 2. The method of claim 1,further comprising co-administering forskolin, colforsin, or a dopamineagonist to the subject.
 3. The method of claim 2, wherein the dopamineagonist is L-DOPA.
 4. The method of claim 1, wherein the compound hasformula:

or any pharmaceutically acceptable salt, hydrate, solvate, ester,stereoisomer mixture, or enantiomer thereof.
 5. The method of claim 1,wherein the compound has formula:

or any pharmaceutically acceptable salt, hydrate, solvate, ester,stereoisomer mixture, or enantiomer thereof.
 6. The method of claim 1,wherein the compound has formula:

or any pharmaceutically acceptable salt, hydrate, solvate, ester,stereoisomer mixture, or enantiomer thereof.
 7. The method of claim 1,wherein the compound has formula:

or any pharmaceutically acceptable salt, hydrate, solvate, ester,stereoisomer mixture, or enantiomer thereof.
 8. The method of claim 1,wherein the compound has formula:

or any pharmaceutically acceptable salt, hydrate, solvate, ester,stereoisomer mixture, or enantiomer thereof.